Modulators of Itch ubiquitinase activity

ABSTRACT

The present invention relates to the identification of new drug targets for therapy of disorders including cancer. In particular, the present invention relates to inhibition of the E3 ubiquitin ligase, Itch, as a means for treating disorders. Furthermore, the present invention relates to the regulation of p63 and p73 stability in cells. In particular, the invention relates to the modulation of the regulation of p63 and p73 stability in cells through modulation of the expression or activity of Itch. Moreover, the invention relates to the use of Itch as a target for the development of agents capable of modulating p63 or p73 stability and especially agents capable of modulating the interaction of Itch and pq63 or p73. Such agents may be useful in therapeutic applications including cancer treatment and modulation of skin differentiation.

This application is a U.S. National Phase Application pursuant to 35U.S.C. 371 of International Application No. PCT/GB2006/000181, which wasfiled Jan. 19, 2006, claiming benefit of priority of Great BritainPatent Application No. 0501202.6, which was filed Jan. 20, 2005 and U.S.Provisional Patent Application No. 60/646,425, which was filed Jan. 24,2005. The entire disclosure of each of the foregoing applications isincorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention relates to the identification of new drug targetsfor therapy of disorders including cancer. In particular, the presentinvention relates to inhibition of the E3 ubiquitin ligase, Itch, as ameans for treating disorders. Furthermore, the present invention relatesto the regulation of p63 and p73 stability in cells. In particular, theinvention relates to the modulation of the regulation of p63 and p73stability in cells through modulation of the expression or activity ofItch. Moreover, the invention relates to the use of Itch as a target forthe development of agents capable of modulating p63 or p73 stability andespecially agents capable of modulating the interaction of Itch and p63or p73. Such agents may be useful in therapeutic applications includingcancer treatment and modulation of skin differentiation.

BACKGROUND OF THE INVENTION

Although our understanding of the mechanisms and possible treatment ofcancer has increased over recent years, cancer remains a major cause ofdeath throughout the developed world. Non-specific approaches to cancermanagement, such as surgery, radiotherapy and generalized chemotherapy,have been successful in the management of some circulating andslow-growing solid cancers. However, many types of cancer are generallyhighly resistant to standard treatments. Accordingly, there is a needfor further, and more effective, cancer therapies.

The development of new therapies depends on the identification ofsuitable targets for drug activity.

The tumour suppressor gene p53 induces cell cycle arrest and promotesapoptosis thereby preventing transformation of cells. Inactivation ofthe tumour suppressor gene p53 is the most common genetic defect incancer affecting more than half of all human tumours. The p53 protein isstabilized in response to genotoxic stress, metabolic changes and otherpotentially dangerous events which can result in transformation ofcells.

p63 and p73 are members of the p53 family of transcription factors andhave been shown to act in a pathway parallel to that of p53, beingup-regulated in response to DNA damage and inducing growth arrest andapoptosis in a p53 independent pathway. Regulation and function of p63and p73 is reviewed in Melino et al. 2003. They both induce cell cyclearrest and apoptosis and have been recently shown to act as tumoursuppressors in vivo (Flores et al., 2005).

The importance of p63 and p73 in tumour suppression is demonstrated bythe finding that disruption of p63 and p73 in p53−/− cells increasestheir transformation capacity. Recently it has been shown that thespecific p53 mutations commonly found in cancers such as Li Fraumenisyndrome lead to functional inactivation of p73 and p63 and thereforeinactivation of p63 and p73 is relevant to in vivo tumorigenesis (Langet al, 2004; Olive et al, 2004).

Both p63 and p73, like p53, have a modular structure (FIG. 1A) (Kaghad,1997). They share a high degree of sequence homology with p53 and canbind to p53-responsive elements activating the transcription of p53target genes, such as those inducing cell cycle arrest and promotingapoptosis (Catani, 2002; De Laurenzi, 1998; De Laurenzi, 2000).

Unlike p53, however, p73 and p63 are expressed as different isoforms(Kaghad, 1997; Ueda, 1999) some of which lack the transactivation domainand are believed to act as dominant negative proteins (Melino, G etal.). Most of the variation generated by alternative splicing occurs atthe 3′ end, in a part of the sequence that does not have a counterpartin p53. The existence of these variant isoforms has made it difficult todetermine the importance of p63 and p73 in tumour suppression. However,recent work using p63/p73 mutant mice has clearly demonstrated that boththese proteins have tumour suppressor functions independent of p53. Inaddition, p63 and p73 mutant mice are predisposed to aggressiveepithelial tumours common in humans (e.g. lung and mammaryadenocarcinomas), unlike p53 mutant mice, which primarily develop thymiclymphomas and sarcomas (Flores et al, 2005).

At least six different p73 proteins (

to η) are generated (De Laurenzi, 1999; De Laurenzi, 1998; Ueda, 1999)while at least three different p63 proteins are generated asalternatively spliced C-terminal isomeric forms. In addition, both p63and p73 genes exploit an alternative promoter and an extra exon (exon3′) to generate N-terminally truncated isoforms (ΔNp63 and ΔNp73). Thesevariants lack the transactivation domain and act as “dominantnegatives”, blocking the function of either p53, p63 or p73 full-lengthproteins (Grob, 2001; Sayan et al., 2004; Yang, 2000). The relativelevels of TA and ΔN isoforms determine cell fate, resulting in eithergrowth arrest and death or uncontrolled proliferation.

TAp73 steady state protein levels are up-regulated in response to DNAdamage in a fashion distinct from p53 (Agami, 1999; Gong, 1999; Yuan,1999) while ΔNp73 is rapidly degraded (Maisse et al., 2004). ΔNp63expression is transcriptionally reduced by p53 suggesting that it doesnot inhibit the tumour suppression activity of p53 and TAp73 in the sameway. These observations suggest an important differential role for theseisomers in carcinogenesis (Melino, 2002; Melino et al., 2003; Stiewe,2002; Zaika, 2002).

The role of p63 and p73 in cell cycle and apoptosis suggests that theirmodification can contribute to enhanced cell death in tumours. Inaddition, several mutations in p63 are associated with genetic epidermalsyndromes while p73 overexpression is sufficient to trigger neuronaldifferentiation. Accordingly, modification of p63 or p73 stability maybe a therapeutic strategy in the treatment of cancer and/ordevelopmental disorders.

While ubiquitination and proteasomal-dependent degradation of p53 isregulated by its transcriptional target MDM2, the regulation of p73 andp63 protein degradation is controlled by distinct E3 ligases. To datevery little is known of the molecular mechanisms underlying theregulation of p63 and p73 protein steady state levels and theirmodulation as a possible therapeutic strategy has not been fullyexplored. While some approaches have sought to use proteosome inhibitorsto inhibit the degradation of p53-family proteins and thus induceapoptosis, in clinical trials these inhibitors have been found to havevery little specificity and lead to up-regulation of a large number ofproteins. Moreover, while a number of cancer therapeutic strategies havetargeted p53, more than 50% of tumours are p53 deficient. Accordingly,there is a need for therapeutics that can target p53 independentpathways.

SUMMARY OF THE INVENTION

The present invention identifies that a modulation of Itch activity canprovide an important cellular response for therapy of various disorders.In particular, the present invention demonstrates that an inhibition ofItch activity regulates apoptosis in a cell.

Accordingly, in a first aspect of the invention, there is provided amethod of modulating apoptosis in a cell comprising the step ofdecreasing or otherwise altering the functional activity of Itchpolypeptide or the nucleic acid encoding it. The changes in Itchactivity results in an altered stability of p63 and p73 proteins, aswell as their activity, such as for example their ability to regulateapoptosis. Suitably, said modulation of apoptosis confers death in acell.

Suitably, the invention provides a method of inducing apoptosis in acell by inhibiting Itch.

Preferably, Itch inhibition is through inhibition of Itch activity. Inanother embodiment, Itch inhibition is through inhibition of Itch mRNAexpression through techniques such as RNAi.

In addition, the present invention identifies that the inhibition ofItch activity sensitises cells to cell death induced by cytotoxic agentssuch as DNA damaging agents. A large number of DNA damaging agents willbe familiar to those skilled in the art. In particular, treatment withDNA damaging compounds (such as, for example, cisplatin, etoposide,doxorubicin, gamma radiation, campodacin, taxol) as well as treatmentwith UV results in rapid downregulation of Itch. Accordingly, thepresent invention provides a method for sensitising cells to DNAdamaging agents through inhibition of Itch activity. Such methods can beuseful in the treatment of cancer.

Accordingly, in a further aspect of the invention there is provided amethod of treating cancer comprising administering an agent whichinhibits Itch activity simultaneously or sequentially with a DNAdamaging agent. Suitable DNA damaging agents include cisplatin,etoposide, doxorubicin, gamma radiation, campodacin, taxol as well astreatment with UV.

Suitably said method comprises the steps of treating a cell with an RNAimolecule capable of interfering with Itch mRNA such that Itch expressionis decreased and treating the cells with a DNA damaging agent. SuitableRNAi molecules are disclosed herein.

The present invention further identifies an interaction between Itch, aHect ubiquitin-protein ligase, and the proteins p63 and p73. Inparticular, the description herein demonstrates that Itch ubiquitinatesp73 targetting it for proteosomal destruction. Moreover, it has beendetermined that Itch selectively binds and ubiquitinates p73 but notp53. Prior to the present description, very little was known about themolecular mechanisms underlying the regulation of p73 protein steadystate levels. MDM2, the E3 ubiquitin ligase that regulates thedegradation of the cognate protein p53 via a proteasomal dependentpathway, binds to p73 but does not promote its degradation (Balint,1999; Dobbelstein, 1999; Lohrum, 1999; Ongkeko, 1999; Zeng, 1999).

Itch ubiquitinates upon binding. In particular, Itch binds andubiquitinates proteins including the TA and ΔN isoforms of p73.Ubiquitination determines proteasome-dependent degradation of proteins.Where TA and ΔN isoforms are ubiquitinated by Itch, they are targetedfor destruction leading to reduced levels of the proteins in the celland therefore decreasing cell cycle arrest and apoptosis. Conversely,where ubiquitination of these proteins is reduced by inhibiting Itchactivity, the level of protein in the cell increases therefore enhancingcell cycle arrest and apoptosis. Itch is therefore a drug target for theregulation of this pathway and the treatment of, inter alia, conditionssuch as cancer where enhanced apoptosis is desirable.

Accordingly, the present invention identifies that down-regulation ofItch activity leads to an increased level of regulated proteins,including p73, which results in increased cell cycle arrest andapoptosis. In addition, it is shown that, upon DNA damage, Itch itselfis down-regulated, allowing p73 protein levels to rise and thusinterfere with p73 function.

In another aspect of the present invention, therefore, there is provideda method of modulating p63 or p73 stability in a cell comprisingmodulating Itch activity or expression.

As shown herein, Itch binds to ΔNp63, TAp73, ΔNp73 and to a lesserextent TAp63. Accordingly, one suitable method of modulating Itchactivity is to modify the binding of Itch to ΔNp63, TAp73 and ΔNp73.Moreover, it is demonstrated herein that Itch binding to p63 and p73occurs between the PY motif of p63 and p73 and the WW domain of Itch.Accordingly, one suitable method for modulating Itch activity is todisrupt binding between PY and WW.

In one embodiment, the method of modulating p63 or p73 stability may bethrough modulating Itch expression. Suitable methods of modulating geneexpression will be familiar to those skilled in the art. Such methodsinclude, for example, RNAi. Suitable RNAi molecules and methods formodulating expression of Itch are described herein. RNAi methods resultin decreased levels of expression of the target protein. RNAi inhibitionof Itch expression has been demonstrated herein to downregulate Itchexpression and result in stabilisation of its substrates including TAand ΔN p73 which, in turn, leads to increased levels of p73.

Conversely, Itch over-expression can be used to promote decreasedstability of its substrates, including TA and ΔN p73, leading to theirrapid degradation.

Accordingly in another aspect of the invention there is provided RNAimolecules for use in a method of modulating p63 or p73 stability.

DNA damage through agents such as cisplatin, etoposide, doxorubicin,gamma radiation, campodacin, taxol, as well as treatment with UV resultsin rapid downregulation of Itch which correlates with increases in p73levels. This increase in p73 levels is thought to be through proteinstabilisation. The invention further provides an assay method foridentifying one or more agents that modulate the activity and/orexpression of Itch.

Suitably such assay methods enable agents which modify the functionalactivity of Itch by modifying its ubiquitinase activity to beidentified. Suitable assays for measuring ubiquitination of a substrateare described herein.

Accordingly, there is provided a method for identifying an agent whichmodulates Itch activity comprising:

incubating an agent or agents to be tested with an Itch molecule in thepresence of a reconstituted in vitro ubiquitination system,

determining the amount of ubiquitin ligated to Itch in the presence ofthe agent or agents to be tested; and

selecting those agents which modulate the amount of ubiquitin ligated toItch compared to the amount of ubiquitin ligated to Itch in the absenceof the agent or agents to be tested.

Suitably, an agent identified as a modulator of Itch activity can befurther assayed to determine its interaction with p63/p73. Inparticular, such assays may identify those agents which modulate thelevels or activity of p63 or p73. Suitable assays for determining saidinteraction include cell based assays in which the candidate agent isincubated in cells and the expression levels of p63/p73 is determined byWestern Blot analysis, for example.

There is also provided a method for identifying an agent which modulatesItch activity on its substrates, comprising: incubating an agent oragents to be tested with an Itch molecule and a ΔNp63, TAp73 or a ΔNp73molecule in the presence of a reconstituted in vitro ubiquitinationsystem, determining the presence of ubiquitinated forms of ΔNp63, TAp73or a ΔNp73 in the presence of the agent or agents to be tested; andselecting those agents which modulate the amount of ubiquitinated formsof ΔNp63, TAp73 or a ΔNp73 compared to the amount of said ubiquitinatedforms obtained in the absence of the agent or agents to be tested.

By a reconstituted in vitro ubiquitination system, is meant a selectionof reagents which enable a ubiquitination reaction to occur. Suchreagents include, for example ubiquitin, an ATP source, E2, E1 and soforth. Examples of suitable systems are described herein.

Ubiquitinated forms of a protein may be detectable by measuring thepresence of higher molecular weight forms of ΔNp63, TAp73 or ΔNp73proteins. Alternative methods for detecting the ubiquitination ofproteins are familiar to those skilled in the art. Suitable methods aredescribed, for example, in WO 01/75145.

In another aspect, the invention provides a method for identifying oneor more agents capable of modulating the stability of p63 or p73,comprising the steps of:

(a) incubating an Itch molecule with the agent or agents to be assessed;and

(b) identifying those agents which influence the binding of Itch to p63or p73.

Preferably, the agent or agents bind to the Itch molecule and/or the p63or p73 molecule and, in particular, a ΔNp63, TAp73 or a ΔNp73 molecule.

Accordingly, there is also provided a method for identifying a leadagent for a pharmaceutical useful in the treatment of disease,comprising: incubating an agent or agents to be tested with an Itchmolecule and a ΔNp63, TAp73 or a ΔNp73 molecule, under conditions inwhich, but for the presence of the agent or agents to be tested, Itchand ΔNp63, TAp73 or a ΔNp73 form a complex with a reference bindingaffinity;

determining the binding affinity of the complex of Itch and ΔNp63, TAp73or a ΔNp73 in the presence of the agent or agents to be tested; and

selecting those agents which modulate the binding affinity of thecomplex with respect to the reference binding affinity.

In one embodiment of the methods described herein, whole molecules maybe used. In another embodiment, the binding parts of the molecules maybe used. As set out herein, the WW domains of Itch are involved inbinding ΔNp63, TAp73 or ΔNp73. Similarly the PPxY motif is the regionwithin ΔNp63, TAp73 or ΔNp73 that interacts with the WW domain.Accordingly, the method of the invention may utilise fragments of themolecules comprising the appropriate binding domains.

In another aspect, the method for identifying agents which modulate thefunctional activity of Itch by modulating its ubiquitinase activity canbe performed in vivo in a cell. In cell based assays, interactions maybe measured in a relevant environment. Such a method suitably comprises:transfecting cells with expression systems to ensure expression of Itchand ΔNp63, TAp73 or ΔNp73 and ubiquitin and subsequently determining thepresence of ubiquitin-ΔNp63, TAp73 or ΔNp73 conjugates. Suitable methodsare disclosed herein. Molecular interactions are detectable, forexample, by two-hybrid screens, in which a gene expressing a detectablemarker is placed under the control of a promoter which is responsive toa transcription factor assembled by the interaction of the two moleculesunder test. Other assays may be used to detect molecular interactions,for example co-immunoprecipitation from transfected Hek293 cell lysates.

In a further aspect, the invention relates to an agent identifiable bythe method of any aspect of the invention, capable of modulating thebinding of Itch to ΔNp63, TAp73 or ΔNp73, modulating the ubiquitinaseactivity of Itch or modulating the stability of ΔNp63, TAp73 or ΔNp73.For example, such an agent may be small molecule inhibitor, an antibody,which is preferably specific for Itch, a polypeptide, such as apolypeptide aptamer, or an Itch molecule such as a dominant negativemutant of Itch. Other suitable agents are described herein.

In addition, mutants of both binding the PY motif of p73 and the WWdomain of Itch have been generated and result in loss of binding andubiquitination of p73. Accordingly, other suitable agents include thesemutant molecules.

In addition, the invention provides research tools comprising modifiedcell lines which enable substances to be tested for their ability tomodulate the activity of Itch on p73.

The invention moreover provides a method for modulating the activity ofItch in a cell, comprising administering to the cell an agent as setforth above, as well as a pharmaceutical composition comprising, asactive ingredient, a therapeutically effective amount of such and to amethod for treating a condition associated with cell proliferation,comprising administering to a subject a therapeutically effective amountof said agent.

Preferably, the disease is a disease involving uncontrolled cellproliferation.

In a further aspect, the present invention provides a method of treatinga cancer in a patient, said method comprising the step of administeringto said patient a therapeutically effective amount of an agent that iscapable of modulating the p73 ubiquitination activity of Itch.

p73 is a critical regulator of the response to chemotherapy in severaltumour cell lines (Irwin et al., 2003). This implies that treatment ofchemotherapy-resistant tumours with Itch inhibitors would result inaugmented p73 expression levels and consequent sensitization tochemotherapy. Accordingly, in one embodiment, said cancer is achemotherapy resistant tumour.

Itch is activated by JNKs to induce the degradation of c-Jun. Both JNKand c-Jun are involved in the regulation of the cellular response to DNAdamage. Therefore, inhibition of Itch would have an impact on how cellsrespond to DNA damage. In particular, it could result in c-Jun (and p73)dependent sensitization to the cytotoxic effects of chemotherapeuticagents.

In particular, it is shown herein that Itch selectively binds andubiquitinates p73 but not p53. Accordingly, the method of the presentinvention is particularly suitable for treating cells in a p53independent manner. This is particularly relevant in the context ofenhancing the response to chemotherapy in resistant tumours containingmutant p53. As p53 is inactivated in >50% tumours, activation of p73 intumour cells is proposed as a good mechanism of targeting tumour cells.

Up-regulation of TAp73 by down-regulation of Itch results in increasedin vivo sensitivity to drugs. We therefore propose that pharmacologicalinhibition of Itch can be used to sensitize tumour cells to treatment.The effect of Itch on other targets such as c-Jun might contribute tothe final effect on DNA damage-dependent apoptosis. In addition aspecific Itch inhibitor can be an extremely valuable tool for scientistin the field.

p63 is expressed in the staminal cell compartment of the epidermis, andits mouse knockout results in the total absence of epidermis. Inaddition, altered expression of p63 proteins have been identified inskin cancers and a deregulation of p63 in skin diseases (psoriasis,ichthyosis) has been shown.

Mutations in p63 are associated with a number of developmentalabnormalities including a combination of hand and foot anomalies andmammary gland aplasia or hypoplasia which characterize the followingsyndromes: ectrodactyl), ectodermal dysplasia, clefting (EEC) syndrome,ulnar-mammary syndrome, limb-mammary syndrome (LMS), ankyloblepharon,ectodermal dysplasia, clefting (AEC) syndrome, nonsyndromic splithand/foot malformation (SHFM) and acro-dermato-ungual-lacrimal-tooth(ADULT) syndrome.

In particular, mutations in the p63 gene in 3q27 have been detected inpatients with EEC syndrome, in nonsyndromic split hand/foot malformation(SHFM) and subsequently in ankyloblepharon, ectodermal dysplasia,clefting (AEC) syndrome, in ADULT syndrome, and in LMS. These aredescribed, for example, in Han G. Brunner, Ben C. J. Hamel, Hans vanBokhoven. American Journal of Medical Genetics. Volume 112, Issue 3,Pages 284-290)

More recently, additional mutations were found in another syndromecharacterized by similar clinical characteristics, the Rapp Hodgkinsyndrome. Other syndromes, such as the lacrimo-anriculo-dento-digital(LADD) syndrome, the ectrodactyly cleft palate (ECP) syndrome, therecessive Bowen-Armstrong and the curly hair ankyloblepharon naildysplasia syndrome (CHANDS) syndromes may also be associated with p63mutations.

Other skin diseases include acute or chronic skin diseases such aspsoriasis, icthyosis, seborrheic keratoses and Bowens lesions. Increasedexpression of p63 has been identified for seborrheic keratoses andBowens lesions. While this was mostly restricted to the basal layer,significant diffuse staining was also noted. Moreover, p63 expression isincreased in squamous cell carcinomas.

Accordingly, in another aspect, the present invention provides a methodof treating epidermal development disorders or skin diseases in apatient, said method comprising the step of administering to saidpatient a therapeutically effective amount of an agent that is capableof modulating the p63 ubiquitination activity of Itch.

In one embodiment, there is provided a method of treating a patient soas to regenerate the skin where it is severely burnt.

Molecules able to regulate the enzymatic activity of Itch might have apotential use in epidermis. This is based on two findings: (i) Itch ishighly expressed in epidermis, (ii) Itch regulates theubiquitinin-dependent degradation of TAp63 and ΔNp63, two proteins thatare crucial for skin development and skin homeostasis, as describedherein.

Consequently, molecules that regulate (inhibition or induction) of Itchactivity, will have a profound effect on the skin and may affectepidermal homeostasis by increasing/reducing the proliferation status.This will predict a potential use both in psoriasis and ichthyosis andallow better in vitro growth and terminal differentiation ofkeratinocytes and artificial skin.

Accordingly, in a further aspect there is provided a method ofincreasing sensitivity of a tumour cell to a chemotherapeutic agentcomprising reducing Itch activity, expression levels or function.Suitably, reduction of Itch is by RNAi. In another embodiment, Itchactivity on p73 is through disrupting the association through the WWdomains.

Specific antibodies for Itch could be used to predict tumours withdifferent chemosensitivity, according to the ability of Itch to regulatep63/p73 mediated DNA damage and chemosensitivity. Specific methods toidentify mRNA of Itch, such as PCR procedures, could be used for thesame purpose. The relative expression of Itch and its substrates (TAp73,ΔNp73, ΔNp63) could be used as a chemosensitivity predictor to tailorindividual cancer chemotherapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 p73 binds to Itch.

(A) Schematic representation of the modular structure of the p73α, p73δ,p53 and Itch proteins. The main structural domains are indicated:transactivation domain (TA), DNA binding domain (DBD), oligomerizationdomain (OD), sterile alpha motif (SAM), amino-terminal C2 domain (C2),WW domains (WW), carboxyl-terminal Hect domain (HECTc). The p73 regionfrom Met452 to Ala489, containing the PPxY motif, used as bait in thephage library panning experiment, is also shown. Bars indicate theregions of Itch expressed by the clones selected in the screening.

(B) T7 plaques from the unselected library and from the enriched phagepool, transferred to a cellulose membrane and probed with GST-PY areshown. PCR demonstrating the enrichment of clones containing the Itch WWdomains is shown in the lower panel.

(C) GST pull down. Hek293 cells were transfected with HA TAp73

(TAp73α) or empty vector (pCDNA) and lysates were incubated with GSTalone, or a GST fusion protein containing the four WW domains of Itch(GST-WW). The retained proteins were detected with anti-p73 antibody(upper panel). The same blot was reprobed with anti-GST polyclonal serum(lower panel). Coimmunoprecipitation of overexpressed proteins: HA-TAp73

or HA TAp53 (D) or HA-ΔNp73

(E) or TAp73αY487F, TAp73αY407F and TAp73αY407F/Y487F (F) weretransiently transfected in Hek293 cells with either Myc-Itch or Myc-ItchMUT expression vectors. Cells were treated with or without MG132 beforelysis. Cell extracts were IP with anti-Myc antibody. The immunecomplexes were subjected to Western-blot analysis with anti-HA antibody(upper panels). The same blots were re-probed with anti-Myc antibody(middle panels). Aliquots of total cell extracts from unprocessed cells(25 μg/lane) were directly subjected to immuno-blot analysis with antiHA antibody (lower panels).

(G) Co-IP of endogenous p73 and Itch proteins. Cells were IP withantibodies against either p73 (mix of clones C17 and C20, Santa Cruz) orp53 (mix of clones D01 and 1801 Santa Cruz) and Western blot wasperformed with antibody against: Itch, p73, p53 and Actin. As a control,IP was performed also with an anti-Actin antibody.

FIG. 2 Itch ubiquitinates p73. (A) TAp73

and TAp73δ and p53 proteins were in vitro translated in the presence of[³⁵S] methionine and incubated with purified Itch (GST-Itch) or itscatalytically inactive mutant (GST-Itch MUT) in the presence of ATP,ubiquitin, and bacterially expressed E1 and E2 (UbcH7). Lanes 10 to 12show in vitro translation with the empty vector (pCDNA). Lanes 13 to 16show an aliquot ( 1/10) of the in vitro translated proteins used in theubiquitination reaction. To demonstrate that p73 ubiquitination couldalso occur in a more physiological system, Hek293 cells were transientlyco-transfected with expression plasmids for HA tagged ubiquitin (Ub-HA),Flag-TAp73

or Flag-p53 (B) Flag-ΔNp73

(C) or Flag-TAp73αY487F, Flag-TAp73αY407F and Flag-TAp73αY407F/Y487F (D)and Myc-Itch or Myc-Itch MUT. 48 hours after transfection cells weretreated with MG132 and then collected. Lysates were subjected to IPusing an anti-Flag antibody. Immune complexes were revealed with anti-HAantibody (upper panels). No Ub-HA-conjugates are present when Ub-HA isomitted from the reaction (B-lanes 10-18). The p73 and p53 proteinexpression levels are demonstrated by probing the same membranes withanti-Flag antibodies (middle panels) and those of Itch by probing thesame membrane with anti-Myc antibodies (lower panels).

FIG. 3 Effect of Itch expression on the steady-state levels of p73. (A)Hek293 cells were transfected with either HA-TAp73

and p53 or HA-TAp73

(A) or Flag-TAp73αY487F, Flag-TAp73αY407F and Flag-TAp73αY407F/Y487F (B)together with either Myc-Itch or Myc-Itch MUT. 48 hours aftertransfection, cells were treated or not with MG132. Equal amounts oftotal protein cell lysates were subjected to Western blotting analysisusing anti-HA antibody or anti Flag antibody (upper rows) to detect thesteady state levels of p73s and p53 proteins. The same blots werere-probed with anti-Myc antibody in order to detect the expressionlevels of Itch (middle rows) and with anti-Actin antibody to show equalloading (lower rows). ³⁵S pulse-chase. H1299 cells were transfected withHA-TAp73

(C) or HA-ΔNp73

(D) together with Myc-Itch or pCDNA-Myc. 48 hours post-transfectioncells were labelled with 250 μCi/ml of Redivue PRO-MIX (L-[³⁵S] in vitrocell labelling mix). Unlabeled Met and Cys (1 mg/ml) were added andcells were collected at the indicated time points. Immunoprecipitationwere performed with anti-HA (Y-11) polyclonal antibody.Immunoprecipitates were washed and run on a SDS-PAGE and detected byautoradiography. For cycloheximide blocking experiments Hek293 cellswere transfected with either HATAp73

(E), HA-ΔNp73

(F), or HA-p53 (G) together with either Myc-Itch or pCDNAMyc. 24 hoursafter transfection cells were treated with cycloheximide and collectedat different time points. Equal amounts of total protein cell lysateswere subjected to Western blotting analysis using anti-HA antibody. Todemonstrate equal loading the same blots were stripped and re-probedwith anti-β-Tubulin or anti-Hsp-70 antibodies for p73s and p53 blotsrespectively. (C) Western blots were subjected to densitometric analysisand results were normalized based on β-Tubulin or Hsp-70 expressionlevels respectively and reported in graphical form (lower panels).

FIG. 4 Effects of Itch downregulation on p73 protein levels.

(A) Saos-2-TAp73

inducible cells were transfected with siRNA oligonucleotides targettingthe Itch sequence or with a scrambled oligonucleotide. 48 hours latercells were induced to express TAp73

for the indicated time points with doxycycline (inducer). p73 levelsincrease more rapidly and reach higher levels when Itch is downregulated. The lower panel shows endogenous Itch levels. Graphs showdensitometric analysis of the p73 western blots normalized forβ-Tubulin.

(B) Saos-2-TAp73

inducible cells were transfected with oligos targetting the Itchsequence or with a scrambled oligo. Cells were induced to express TAp73

for 14 hours with doxycycline, the inducer was removed and cellscollected at the indicated time points. p73 levels decay more rapidly incells transfected with the scrambled oligo compared to those in whichItch is down regulated. The lower panel shows endogenous Itch levels.Graphs show densitometric analysis of the p73 Western blots normalizedfor β-Tubulin.

(C) Saos-2 cells transfected with oligos targeting the Itch sequence orwith a scrambled oligo and collected 48 hours later. Itchdown-regulation (lower panel) results in an increase of TA and ΔN p73alevels (upper panels).

(D) Western blot for endogenous p73 of wild type MEFs (MEF+/+),non-agouti-lethal 18H Itch deficient MEFs (MEF Itch−/−) and aspontaneously immortalized clone of these MEFs (MEF Itch−/−Immortalized). ΔNp73 levels (the only form detectable in these cells)are higher in MEFs Itch−/−. (E) Immortalized MEFs Itch−/− weretransfected with Myc-Itch WT and collected 48 hours later.Re-introduction of Itch results in ΔNp73 downregulation.

FIG. 5 Nedd4 binds p73 but fails to ubiquitinate it.

(A) Hek293 cells were transfected with HATAp73

HA-ΔNp73

HA-p53 or an empty vector together with Myc-Itch or Myc-Nedd4 in thepresence of MG132. Cells were subjected to IP using anti-HA antibody andanalysed by western blot using an antibody against Myc. Itch and Nedd4co-IP with p73.

(B) Hek293 cells were transfected with HA-TAp73

HA-ΔNp73

or HA-p53 together with Myc-Itch or Myc-Nedd4. Cell extracts wereanalysed by western blot using an antibody against HA. Expression ofItch but not of Nedd4 results in TA and ΔN p73 down-regulation. (C)Cells transfected with the indicated plasmids together with a plasmidexpressing HA tagged ubiquitin (Ub-HA) were analysed by western blotusing an antibody against HA. Higher molecular bands characteristic ofubiquitination appear when Itch but not Nedd4 is overexpressed.

FIG. 6 Itch expression reduces the transcriptional activity of p73.H1299 cells were transfected with the indicated combinations of plasmids(at the different indicated ratios) encoding TAp73α, together withMyc-Itch WT (Itch wt) or Myc-Itch MUT (Itch Mut), or empty controlvector (pCDNA) together with a Bax-(A-B) or MDM2-(C-D) or p21-(E-F)luciferase reporter plasmid and Renilla luciferase reporter plasmid.Cell extracts were prepared 36 hrs later and luciferase activity wasdetermined. Results are represented as fold induction of luciferaseactivity as compared with the control cells. Histograms show the mean ofthree independent experiments; bars indicate standard deviation. (G)H1299 cells were transfected with HA-TAp73

together with Myc-Itch at the indicated ratios. Equal amounts of cellextracts were subjected to western blot with anti p21 antibody (upperpanel), anti-HA (middle panel) and anti-Myc (lower panel).

FIG. 7 Itch is down regulated by DNA damaging agents. Saos-2 cells weretreated with 2 μM doxorubicin (Doxo) for 24 or 48 hours then collectedby trypsinization. Cells were lysed for western blotting (A) and half ofthe cells were ethanol fixed for apoptosis analysis (B). Western blotswere performed with either anti-Itch (upper panel) or anti-p73 (lowerpanel) antibodies. Blots were stripped and re-probed with anti Lamin Bantibody to show equal loading. Apoptosis was evaluated by flowcytometric analysis of PI stained cells. Non-treated controls (N.T.) areshown. H1299 cells transfected with HA-TAp73

(Con) are loaded as a control for p73 western blot.

(C) Western blot using anti-Itch antibody on Hela, H1299, Hek293 andSaos-2 cells treated for 24 hours with 2 μM doxorubicin. Blots werestripped and re-probed with anti-β-Tubulin antibody to show equalloading.

(D) Western blot using anti-Itch antibody on Saos-2 cells treated for 24and 48 hours with either 15 μM etoposide (Etopo) or 5 μM cisplatin(Cisp). Non-treated control cells are also shown (N.T.). Blots werestripped and re-probed with anti p73 antibody and Lamin B antibody toshow equal loading.

(E) Saos-2-TAp73

(upper panel) or Saos-2-ΔNp73

(lower panel) were induced with doxycycline for 14 hours. Doxycyclinewas then removed and cells were treated with the indicated drugs andanalysed at the indicated time points by western blot using anti-HAantibody. Blots were re-probed with anti-β-Tubulin to show equalloading.

(F) Saos-2-ΔNp73

were transfected with oligos for siRNA against the Itch sequence or witha scrambled sequence and then induced with doxycycline for 14 hours.Doxycycline was then removed and cells were treated with doxorubicin andanalysed at the indicated time points by western blot using anti-HAantibody. Blots were re-probed with anti-β-Tubulin to show equalloading.

FIG. 8 Itch controls p73 levels in resting conditions and in response toDNA damage.

(A) Schematic representation of the functional interaction between p73and Itch. Under nonstressed conditions basal levels of both TAp73 andΔNp73 are kept low; in this situation Itch binds to p73 and promotes itsubiquitination and proteasome dependent degradation.

(B) In response to DNA damage Itch is rapidly degraded, reducing p73turn over. TAp73 levels increase while ΔNp73 remain low due to theactivation of a DNA damage dependent ΔN specific degradation pathway.The final outcome is the induction of cell cycle arrest and apoptosis byp73.

FIG. 9 shows Saos-2 cells were transfected or not with Itch-specificsiRNA oligos and then either treated or not with doxorubicin (2 μM).After 24 hours (bars 1-4) and 48 hours (bars 5-8) cells were trypsinizedand incubated with propidium iodide to quantize cell death. Thehistograms represent the mean results of three independent experiments.

FIG. 10 shows ITCH interacts with p63. 293T cells were transfected withdifferent combinations of expression vectors for Myc-tagged WT ITCH(Myc-ITCH WT), enzymatically inactive ITCH (Myc-ITCH MUT), Flag-taggedTAp63α (Flag-TAp63α) and ΔNp63α (Flag-ΔNp63α). Anti-Mycimmunoprecipitates were probed with anti-Flag antibodies. Top panelshows co-immunoprecipitated proteins. Middle panel shows ITCH levels inthe immunoprecipitates. Lower panel shows p63 levels in the inputlysates. Experiments were carried out in the presence of the proteasomeinhibitor MG132

FIG. 11 shows ITCH induces the ubiquitination of ΔNp63α. 293T cells weretransfected with vectors expressing Myc-tagged WT ITCH (Myc-ITCH WT),enzymatically inactive ITCH (Myc-ITCH MUT), Flag-tagged TAp63α(Flag-TAp63α), ΔNp63α (Flag-ΔNp63α) and HA-tagged ubiquitin (Ub HA).Lysates were immunoprecipitated with anti-Flag antibody and probed withanti-HA antibody. Top panel shows ubiquitin-conjugated p63 proteins.Middle panel shows p63 levels in the immunoprecipitates. Lower panelshows ITCH levels in the input lysates. Experiments were carried out inthe presence of the proteasome inhibitor MG132.

FIG. 12 shows ITCH over-expression causes ΔNp63α downregulation. 293Tcells were transfected with vectors expressing Myc-tagged WT ITCH(Myc-ITCH WT), enzymatically inactive ITCH (Myc-ITCH MUT), Flag-taggedTAp63α (Flag-TAp63α) and ΔNp63α (Flag-ΔNp63α) and analyzed for p63steady state levels using anti-Flag antibody (upper panel). Middle panelshows ITCH levels, while lower panel shows tubulin levels as loadingcontrol.

FIG. 13 shows ITCH selectively regulates the half-life of ΔNp63α. (A)293T cells were transfected with Flag-tagged TAp63α expression vector inthe presence of either control vector (top panel) or Myc-tagged WT ITCH(Myc-ITCH WT, lower panel) expression vector. 24 hours aftertransfection cells were incubated with cycloheximide and at theindicated different time points were harvested, lysed and analyzed forp63 levels using an anti-Flag antibody. (B) 293T cells were transfectedwith Flag-tagged ΔN p63α expression vector in the presence of eithercontrol vector (top panel) or Myc-tagged WT ITCH (Myc-ITCH WT, lowerpanel) expression vector. 24 hours after transfection cells wereincubated with cycloheximide (20 μg/ml) and at the indicated differenttime points were harvested, lysed and analyzed for p63 levels using ananti-Flag antibody. In all cases, Tubulin levels were measured asloading control.

FIG. 14 (A) Western blot of 12 cell lines showing Itch expression (B)effect of shRNA (oligonucleotides no. 2 and 18) on Itch levels in (i)cos1, (ii) h1299, (iii) HeLa, and (iv) HEK293T 1=untransfected cells;2=cells transfected with scrambled RNA; 3=cells transfected with shRNAclone 2; 4=cells transfected with shRNA clone 18.

FIG. 15 Histograms showing cell cycle of cells transfected withscrambled, or shRNA against Itch 48 hours after transfection, (a) Cos1,(b) H1299, (c) HEK293T

FIG. 16 Cells showing downregulation of Itch in response to transfectionwith shRNA targeted to Itch, and increased p73 levels, (a) Cos1, (b)HEK293T, (c) H1299

FIG. 17—Western blots of samples from H1299 cells, which were eithertransfected with scrambled or shRNA against Itch and then treated for 24and 48 hours with DNA damaging agents. p73 levels in control and treatedcells are shown.

FIG. 18. ELISA assay for self-ubiquitination of Itch. Wild type Itch(E3) or catalytically inactivated Itch containing the point mutationC830A (E3m), were immobilised onto a glutathione-coated microtiter platewells. Uncoated wells received coating buffer alone. Ubiquitinationreactions were performed in the presence or absence of E1, E2, E3(m) orFLAG-ubiquitin as indicated.

FIG. 19. Graphical representation of the data obtained from a pilotscreen of 1,280 small molecules from the LOPAC¹²⁸⁰™ library in the highthroughput ELISA. Hits were identified from wells showing activity of70% or less of the mean of the positive controls.

DETAILED DESCRIPTION OF THE INVENTION Advantages

Ubiqutin ligases are tractable drug targets that offer great potentialfor drug design. Firstly, they are enzymes and therefore represent easymolecules to target for design of small molecule inhibitors. Inaddition, small molecules that target enzymes which, in turn, regulatedegradation can be used to increase the activity of a target whereotherwise design of molecules to act directly on that target may not bepossible.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods. See,generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Vol. 194,Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.),McPherson et al., PCR Volume 1, Oxford University Press, (1991), Cultureof Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney.1987. Liss, Inc. New York, N.Y.), and Gene Transfer and ExpressionProtocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc.,Clifton, N.J.). These documents are incorporated herein by reference.

Itch

Ubiquitin is a protein that can become covalently attached to lysineresidues on target proteins. This reaction requires the sequentialaction of 3 enzymes, the E1, E2 and E3 enzymes. After modification witha number of ubiquitin moieties linked through Lys 48 of ubiquitin, theubiquitinated protein is targeted for ATP-dependent degradation by the26S proteasome. In this process, the E3 ubiquitin ligases catalyze thefinal transfer of ubiquitin to a specific substrate, thus governing thespecificity of substrate recognition (Hicke, 2001; Kloetzel, 2001;Weissman, 2001). Therefore, therapeutic targetting of particular E3ligases will produce selective alterations in the degradation rates ofsmall groups of proteins, rather than inhibiting ubiquitination anddegradation of all proteins in the proteasome. E3 enzymes contain twoseparate activities: a ubiquitin ligase activity to conjugate ubiquitinto substrates and form polyubiquitin chains via isopeptide bonds, and atargetting or binding activity to physically bring the ligase andsubstrate together.

In addition to its role in ubiquitination and degradation, ubiquitin canalso confer a subcellular address label on proteins, resulting inchanges to their intracellular localisation, and therefore, function.

Itch is an example of an E3-ubiquitin ligase that belongs to theNedd4-like E3 family, and is characterized by a modular organizationthat includes: an N-terminal protein kinase C-related C2 domain;multiple WW domains; and a C-terminal HECT (homologous to theE6-associated protein carboxyl terminus) Ubiquitin (Ub)-protein ligasedomain (Harvey, 1999) (FIG. 1A). The Itch mouse homologue gene is absentin the non-agouti-lethal 18H (Itchy) mice which display profound immunedefects (Fang, 2002; Perry, 1998).

Itch ubiquitinates Jun-B targetting it for degradation (Fang et al Nat.Immunol. 2002). Itch has been shown to be phosphorylated by JNK-1 andJNK-2 in response to T-cell stimulation (Gao et al Science 2004) andphosphorylation of Itch results in increased catalytic activity (Gao etal Science 2004). In addition, Itch ubiquitinates cJun and Jun-Btargetting them for degradation and modulating cytokine production (Gaoet al Science 2004). Itch also regulates TGF-Beta response by promotingSMAD phosphorylation (Bai et al Mol. Cell. 2004) and regulates Notch(Qui et al J. Biol. Chem. 2000). Itch also undergoes self-ubiquitination(Gao et al Science 2004).

Human Itch is described by Perry W L, Hustad C M, Swing D A, O'SullivanT N, Jenkins N A, Copeland N G. Nat. Genet. 1998 18:143-6 and thesequences deposited under GenBank Accession numbers NM_(—)031483(nucleotide) (gi27477108) and NP_(—)113671 (protein) (gi:27477109).

Reference to Itch herein includes Itch and its variants, homologues,fragments or derivatives.

p63 and p73

Reference to p63 and p73, as used herein, includes p63, p73 and any oftheir variants, homologues, fragments or derivatives including truncatedand alternatively spliced forms or isoforms as described herein. Inparticular, reference to p63 and p73 includes reference to TAp73, ΔNp73and ΔNp63. These isoforms are described, for example, by Melino et al.,Trends in Biochemical Sciences, Vol. 28, No. 12, December 2003, Melinoet al. Nature Reviews, Vol. 2, p. 1-11, August 2002. The mRNA sequenceencoding p63 protein can be found in GenBank NM_(—)003722, while themRNA encoding p73 protein can be found in GenBank NM_(—)005427.

Variants, Homologues, Fragments or Derivatives

The term “variant” is used to mean a naturally occurring polypeptide ornucleotide sequence which differs from the naturally occurring sequence.

The term “fragment” indicates that a polypeptide or nucleotide sequencecomprises a fraction of a sequence. It may comprise one or more largecontiguous sections of sequence or a plurality of small sections. Thesequence may also comprise other elements of sequence, for example, itmay be a fusion protein with another protein. Preferably the sequencecomprises at least 50%, more preferably at least 65%, more preferably atleast 80%, most preferably at least 90% of a sequence described herein.

The term “homologue” means an entity having a certain homology with thesubject amino acid sequences and the subject nucleotide sequences. Here,the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence, which may be at least 70, 75, 80, 85 or 90%identical, preferably at least 95 or 98% identical to the subjectsequence. Typically, the homologues will comprise the same active sitesetc. as the subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

In the present context, a homologous sequence is taken to include anucleotide sequence, which may be at least 70, 75, 80, 85 or 90%identical, preferably at least 95 or 98% identical to the subjectsequence. Typically, the homologues will comprise the same sequencesthat encode for the active sites etc. as the subject sequence. Althoughhomology can also be considered in terms of similarity, in the contextof the present invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons may be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However, for some applications, it is preferred to usethe GCG Bestfit program. A new tool, called BLAST 2 Sequences is alsoavailable for comparing protein and nucleotide sequence (see FEMSMicrobiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1):187-8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix—such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues, which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example, according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) may occur i.e. like-for-like substitution—such as basic forbasic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids—such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids—such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#)*,L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe)—such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid^(#) and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups—such as methyl, ethyl or propyl groups—inaddition to amino acid spacers—such as glycine or β-alanine residues. Afurther form of variation involves the presence of one or more aminoacid residues in peptoid form will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example, Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences may be modified by any methodavailable in the art. Such modifications may be carried out to enhancethe in vivo activity or life span of nucleotide sequences useful in thepresent invention.

Alleles of sequences—such as Itch, p63 or p73—are also included. As usedherein, an “allele” or “allelic sequence” is an alternative form of thereceptor. Alleles result from a mutation, ie., a change in the nucleicacid sequence, and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Preferably, function isnot altered. Any given gene may have none, one or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to deletions, additions or substitutions of amino acids. Eachof these types of changes may occur alone, or in combination with theothers, one or more times in a given sequence.

The term “allele” also includes genetic polymorphisms—such as SNPs(single nucleotide polymorphisms).

The terms “variant”, “homologue”, “derivative” or “fragment” include anysubstitution of, variation of, modification of, replacement of, deletionof or addition of one (or more) nucleic acids from or to the sequenceproviding the resultant nucleotide sequence is capable of coding for aprotein having the desired activity—such as Itch ubiquitin ligaseactivity, preferably being at least as biologically active as theprotein encoded by any one of the sequences shown herein.

Apoptosis

“Apoptosis” or cell death is a controlled intracellular processcharacterised by the condensation and subsequent fragmentation of thecell nucleus during which the plasma membrane remains intact. A cascadeof enzymes including caspases that cleave at aspartic acid residues isactivated in the process.

By “modulating apoptosis” is meant that for a given cell, under certainenvironmental conditions, its normal tendency to undergo apoptosis ischanged compared to an untreated cell. A decreased tendency to apoptosemay also be a measurable increase in cell survival and may be the resultof an inhibition of apoptosis by inhibiting one or more components ofthe apoptotic pathway. An increase in the tendency to undergo apoptosisis measurable by increased cell death, for instance as described below.

Methods for inducing apoptosis are well known in the art and include,without limitation, exposure to chemotherapy or radiotherapy agents andwithdrawal of obligate survival factors (e.g. GM-CSF, NGF) ifapplicable. Differences between treated and untreated cells indicateseffects attributable to the test agent.

Methods for measuring apoptosis are familiar to those skilled in the artand are described herein.

Functional Activity

The “functional activity” of a protein in the context of the presentinvention describes the function the protein performs in its nativeenvironment. Altering or modulating the functional activity of a proteinincludes within its scope increasing, decreasing or otherwise alteringthe native activity of the protein itself. In addition, it also includeswithin its scope increasing or decreasing the level of expression and/oraltering the intracellular distribution of the nucleic acid encoding theprotein, and/or altering the intracellular distribution of the proteinitself.

Expression

The term “expression” refers to the transcription of a genes DNAtemplate to produce the corresponding mRNA and translation of this mRNAto produce the corresponding gene product (i.e., a peptide, polypeptide,or protein). The term “activates gene expression” refers to inducing orincreasing the transcription of a gene in response to a treatment wheresuch induction or increase is compared to the amount of gene expressionin the absence of said treatment. Similarly, the terms “decreases geneexpression” or “down-regulates gene expression” refers to inhibiting orblocking the transcription of a gene in response to a treatment andwhere such decrease or down-regulation is compared to the amount of geneexpression in the absence of said treatment.

Modulating

The term “modulating” may refer to preventing, suppressing, alleviating,restoring, increasing, elevating or otherwise affecting the activityand/or expression of Itch. In particular, the term “modulating” refersto modulating the ubiquitin ligase activity of Itch.

By modulating p63 or p73 stability is meant effecting a change in steadystate protein level of the proteins in the presence of a candidatecompound as compared to those levels in the absence of a candidatecompound. Levels of proteins can be assessed, for example, by Westernblot analysis.

The activity and/or expression of Itch may be modulated by affecting thedegree of binding or interaction between Itch and its substrates such asp63 and p73. By way of example, the degree of binding or interaction maybe increased using an agent that binds to or interacts with amino acidresidues of Itch that increase the activity and/or expression of Itch.By way of further example, the degree of binding or interaction may bedecreased using an agent that binds to or interacts with amino acidresidues of Itch that decrease the activity and/or expression of saidreceptor.

Agents that are able to modulate the activity and/or expression of Itchmay be agonists or antagonists.

Agonists and Antagonists

Agents capable of increasing or elevating the activity and/or expressionof Itch are referred to as agonists.

Agents capable of reducing, inhibiting or blocking the activity and/orexpression of Itch are referred to as antagonists.

Advantageously, the assay methods of the present invention may be usedto identify agents—such as agonists or antagonists—that modulate theactivity and/or expression of Itch.

The term “agonist”, as used in the art, is generally taken to refer to acompound or agent which binds to a protein and increases its activity.The term as used here, however, is intended to refer broadly to anyagent, which increases the activity and/or expression of Itch, notnecessarily by binding to it. Accordingly, it includes agents, whichincrease the expression of one or more proteins, or the biosynthesis ofa molecule, or the expression of modulators of the activity of Itch. Thespecific activity which is increased may be any activity which ischaracteristic of Itch.

The agonist may bind to and compete for one or more sites on Itch.However, the agonist need not necessarily bind directly to the bindingor active site of Itch, and may bind for example to an adjacent site,another protein (for example, a protein which is complexed with Itch) orother entity on or in the cell, so long as its binding increases theexpression and/or activity of Itch.

Increasing the activity of a ubiquitin ligase may also be achieved byincreasing the level of expression of Itch in the cell.

In some embodiments, the present invention relies on blocking ordecreasing the activity of Itch. Agents which are capable of suchactivity are referred to as “Itch antagonists”.

The term “antagonist” is intended to refer broadly to any agent whichinhibits, blocks or decreases the activity or activation of Itch, notnecessarily by binding to it.

Accordingly, it includes agents which affect the expression of Itch, orthe expression of modulators of the activity of Itch. The activity whichis inhibited may be any activity which is characteristic of Itch—such asubiquitin ligase activity. Preferably, the activity, which is inhibited,blocked or decreased, is the ubiquitination of p73 or p63. Morepreferably, the activity, which is inhibited, blocked or decreased isthe ubiquitination and hence proteasomal-dependent degradation of itssubstrates including p73 and p63. Most preferably, the inhibition,blocking or decreasing of Itch ubiquitin ligase activity, mediatesinduction of apoptosis in cells through increased levels of Itchsubstrates such as p73 and p63.

The antagonist may bind to and compete for one or more binding or activesites on Itch. Such binding may block, inhibit or decrease interactionswith Itch substrates. Such binding may block, inhibit or decrease theinteraction between Itch and p73 or p63. However, the antagonist neednot necessarily bind directly to Itch, and may bind for example to anadjacent site, another protein (for example, a protein which iscomplexed with Itch) or any other entity on or in the cell, so long asits binding blocks, inhibits or decreases the activity of Itch.

An antagonist of Itch may include a ligand of Itch, or a fragment ofthis, which is capable of binding to Itch. In addition, whole orfragments of a ligand generated natively or by peptide synthesis may beused to compete with the ligand for binding sites on Itch.Alternatively, or in addition, an immunoglobulin (for example, amonoclonal or polyclonal antibody) capable of binding to Itch may beused. The antagonist may also include a peptide or other small molecule,which is capable of interfering with the binding interaction. Theantagonist may also include an aptamer. Other examples of antagonistsare set forth in greater detail below, and will also be apparent to theskilled person.

Blocking, inhibiting or decreasing the activity of Itch may also beachieved by reducing the level of expression of Itch in the cell. Forexample, the cell may be treated with antisense compounds, siRNA orribozymes for example, having sequences specific to Itch.

In a highly preferred embodiment, the Itch antagonist is a selectiveItch antagonist. As used herein, the term “selective Itch antagonist”means an antagonist having at least 100-fold selectivity over relatedubiquitin ligases including, for example, mdm2, NEDD4 and cCb1.

Advantageously, the Itch antagonist can be used in the treatment ofcancer or epidermal abnormalities. Other disorders include immune systemregulatory disorders.

By way of example only, Itch antagonists may be identified using methodsfor detecting ubiquitin ligase activity such as those described in WO01/75145, for example. Briefly, a candidate antagonist, Itch,preferably, recombinant Itch (or a variant, homologue, fragment orderivative thereof) and an Itch substrate, suitably selected from p73and p63, are configured to permit detection of Itch antagonists bymeasuring incorporation of ubiquitin onto the substrates in the presenceor absence of the candidate antagonist.

Suitably incorporation of ubiquitin is monitored using a system thatproduces a signal which varies with the extent of ubiquitination, suchas the incorporation of ³⁵S methionine in the presence of ATP andubiquitin, as described herein. Other monitoring methods include afluorescence resonance energy transfer system (FRET) as described, forexample, in WO 01/75145.

Another method of screening a library of compounds or agents for Itchantagonists utilises eukaryotic or prokaryotic host cells, which arestably transformed with recombinant DNA molecules expressing a libraryof compounds. Such cells, either in viable or fixed form, can be usedfor standard binding-partner assays. See also Parce et al. (1989)Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA87; 4007-4011, which describe sensitive methods to detect cellularresponses. Competitive assays are particularly useful, where the cellsexpressing the library of compounds are contacted or incubated with alabelled antibody known to bind to Itch—such as ¹²⁵I-antibody, and atest sample such as a candidate compound whose binding affinity to thebinding composition is being measured. The bound and free-labelledbinding partners for the polypeptide are then separated to assess thedegree of binding. The amount of test sample bound is inverselyproportional to the amount of labelled antibody binding to thepolypeptide.

Another technique for screening involves an approach, which provideshigh throughput screening for new compounds having suitable bindingaffinity, e.g., to an Itch polypeptide, and is described in detail in WO84/03564. First, large numbers of different small peptide test compoundsare synthesized on a solid substrate, e.g., plastic pins or some otherappropriate surface; see Fodor et al. (1991). Then all the pins arereacted with solubilized polypeptide and washed. The next step involvesdetecting bound polypeptide. Compounds which interact specifically withthe polypeptide will thus be identified.

Preferably, the methods used to identify Itch antagonists include theadditional step of determining if the Itch antagonist is a selectiveItch antagonist. This optional step comprises measuring the activity ofthe Itch antagonist for activity against another ubiquitin ligase. AItch antagonist with a 100-fold selectivity over another ubiquitinligase is indicative that the Itch antagonist is a selective Itchantagonist.

Specific examples of potential Itch agonists and antagonists includeantibodies or, in some cases, nucleotides and their analogues, includingpurines and purine analogues, oligonucleotides or proteins which areclosely related to the substrates or binding partners of Itch, eg., afragment of the substrate or binding partner.

Subject

The term “subject” includes all animals including humans. Examples ofsubjects include humans, cows, dogs, cats, goats, sheep, horses, andpigs. The mammals to be treated according to this invention are subjectswho have developed proliferative disorders including cancer, immuneregulatory disorders or epidermal abnormalities and/or are sufferingfrom the symptoms associated with disease, or who are at risk fordeveloping the disease, for example having a family history of any ofthese disorders. Those skilled in the art are readily able to identifyindividual patients who are afflicted with such disorders, as well asthose who are susceptible to developing the diseases.

Assays/Methods

Suitable assays for identifying compounds that interact with and/ormodify Itch activity and/or interactions can be formulated.

Such suitable assays include, for example, flourimetric assays forligase activity although other assay readout methods can be used.

One such suitable assay can be a two-step reaction in which HA-taggedp73 or p63 cDNA is cloned into a bacterial expression vector and fusedin frame with GST (C-terminally). A cleavage site for a protease ispresent immediately downstream the cloning site to allow the removal ofthe GST sequence following the last purification step (see Figure A).The p63 (or p73)-GST expression vector may be grown in the BL21 strainand purified using a commercial kit. Preferably, the GST portion of thefusion p63 (or p73)-GST may be removed by incubation with the specificprotease (see Figure A) in order to avoid the GST sequence interferingwith the ubiquitination reaction. Purified HA-E1-coated plates may beincubated with GST-Ubc7-E2 and EGFP-Ubiquitin in the presence of ATP(see Figure A). Subsequently, supernatants from the E1/E2 reactions willbe aliquoted on p63-(or p73-)GST-coated plates in the presence orabsence of Itch (see Figure A). Reactions will be carried out in thepresence or absence of the library. After extensive and stringentwashing, plates will be analyzed by immunofluorescence suitably using anautomated procedure.

Suitable negative controls may be included as follows:

-   -   E1/E2 reactions in the absence of E2    -   E1/E2 reactions in the absence of ATP.    -   E1/E2 reactions in the presence of an EGFP-ubiquitin mutant that        cannot be transferred to E3s.    -   Reactions in the presence of GST only coated plates.

In a further aspect, there is provided a cell-based screening method toidentify agents that modulate Itch activity and/or expression.

Thus, a cell line that expresses Itch may be used to screen for agentsthat modulate Itch activity and/or the expression thereof. For example,screening of peptide libraries or organic libraries made bycombinatorial chemistry with the cell line may be useful foridentification of therapeutic agents that function by modulating Itchactivity and/or expression. Synthetic compounds, natural products, andother sources of potentially biologically active materials can also bescreened using the cell lines in a number of ways deemed to be routineto those of skill in the art.

The agent(s) to be tested, may be administered to the cell in severalways. For example, it may be added directly to the cell culture mediumor injected into the cell. Alternatively, in the case of polypeptideagents, the cell may be transfected with a nucleic acid construct, whichdirects expression of the polypeptide in the cell. Preferably, theexpression of the polypeptide is under the control of a induciblepromoter.

Thus, in a further aspect, there is provided an assay method foridentifying one or more agents that modulate the activity and/orexpression of Itch, the assay method comprising the steps of: (i)providing a cell that expresses or is capable of expressing Itch or avariant, derivative or homologue thereof; (ii) contacting the cell withan agent; and (iii) measuring the levels of ubiquitination of asubstrate such as p63 or p73; wherein a difference between a) levels ofubiquitination in the absence of the agent and b) levels ofubiquitination in the presence of the agent is indicative that the agentcan affect Itch activity and/or expression.

By way of example only, the cell-based screening method to identifyagents that modulate Itch activity and/or expression may be performed asfollows. Cells—such as Hek293 cells—are plated out and grown toconfluence and then washed with, for example, DMEM. DMEM is removed fromthe cells and the agent to be tested or DMEM alone are added (as acontrol). The cells are then incubated with one or more agents. Thecells may additionally be transfected with expression vectors encodingthe Itch substrates p73 or p63. The cells are then incubated and thelevels of substrate ubiquitination determined using Western blotting, asdescribed herein, for example.

Other Potential Assays to Screen for Inhibitors of Itch

Screens for Itch modulators may also be performed by monitoring changesin Itch self-ubiquitination. Suitable assays are described, for example,in the Examples section herein.

A high throughput screen to identify inhibitors of a ubiquitin ligasesuch as Itch, may be configured in a number of ways as understood by theskilled artisan. For example, polyubiquitin chain formation may beassayed using TR-FRET (Hong C A, Swearingen E, Mallari R, Gao X, Cao Z,North A, Young S W, Huang S G. 2003. Development of a high throughputtime-resolved fluorescence resonance energy transfer assay for TRAF6ubiquitin polymerization. Assay Drug Dev Technol. 1:175-80). In thismode, ubiquitin mixtures are optimised to consist of a proportion of twodifferent fluorophore-labelled ubiquitins, which act as a donor andacceptor pair to give a FRET signal when brought together in a polymer.Ubiquitin may be labelled with a FRET partner directly, or indirectlyusing a fluorescently labelled antibody or by labelling the ubiquitinwith biotin in order to combine with avidin or streptavidin bearing aFRET donor or acceptor label. In another variation, the ubiquitinationtarget itself may be labelled with biotin and combined with astreptavidin-labelled fluorophore for use in combination with anappropriate fluorescently-labelled ubiquitin as the FRET partner (YabukiN, Watanabe S, Kudoh T, Nihira S, Miyamato C. 1999. Application ofhomogeneous time-resolved fluorescence, HTRFTM, to monitorpoly-ubiquitination of wild-type p53. Comb Chem High Throughput Screen.2:279-87). This type of assay can be configured without the need forwash stages, however the signal to background ratio for this type ofassay tends to be poorer, and unlike ELISA assays, homogeneous FRETassays are prone to interference from fluorescent library compounds. Theauthors also compare the FRET assay with a scintillation proximity assay(SPA) using I¹²⁵ labelled ubiquitin (I¹²⁵Ub). During the ubiquitinationreaction, I¹²⁵Ub is transferred to the biotinylated p53 substrate, whichin turn is detected by binding to streptavidin-coated PVT SPA beadsfollowed by scintillation counting.

In a modification to the ELISA format, electrochemiluminescence assaysmay be configured to screen for inhibitors of ubiquitin ligases (DavydovI V, Woods D, Safuran Y J, Oberoi P, Fearnhead H O, Fang S, Jensen J P,Weissman A M, Kenten J H, Vousden K H. 2004. Assay for ubiquitin ligaseactivity: high-throughput screen for inhibitors of HDM2. J BiomolScreen. 9:695-703; Kenten J H, Davydov I V, Safuran Y J, Stewart D H,Oberoi P, Biebuyck H A. 2005. Assays for high-throughput screening of e2and e3 ubiquitin ligases. Methods Enzymol. 399:682-701). In this formatthe detection reagent, for example an antibody to polyubiquitin chains,is conjugated to an electroluminescent label such as rutheniumtris-bypyridine or its derivatives. Such a label will emit light underoxidation at an electrode under suitable chemical conditions. For thistype of assay electrodes are incorporated into the base of speciallyconstructed microtiter plates and since the emission only occurs whenthe label is in close proximity to the electrode surface, it is possibleto eliminate or minimise the number of wash steps in the assayprocedure. Such assays also tend to have good sensitivity and dynamicrange.

Agent

As used herein, the term “agent” may be a single entity or it may be acombination of entities.

The agent may be an organic compound or other chemical. The agent may bea compound, which is obtainable from or produced by any suitable source,whether natural or artificial. The agent may be an amino acid molecule,a polypeptide, or a chemical derivative thereof, or a combinationthereof. The agent may even be a polynucleotide molecule—which may be asense or an anti-sense molecule. The agent may even be an antibody.

The agent may be designed or obtained from a library of compounds, whichmay comprise peptides, as well as other compounds, such as small organicmolecules.

By way of example, the agent may be an atom or molecule, wherein amolecule may be inorganic or organic, a biological effector moleculeand/or a nucleic acid encoding an agent such as a biological effectormolecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptidenucleic acid (PNA), a virus, a virus-like particle, a nucleotide, aribonucleotide, a synthetic analogue of a nucleotide, a syntheticanalogue of a ribonucleotide, a modified nucleotide, a modifiedribonucleotide, an amino acid, an amino acid analogue, a modified aminoacid, a modified amino acid analogue, a steroid, a proteoglycan, alipid, a fatty acid and a carbohydrate. An agent may be in solution orin suspension (e.g., in crystalline, colloidal or other particulateform). The agent may be in the form of a monomer, dimer, oligomer, etc,or otherwise in a complex.

The term “agent” is also intended to include, a protein, polypeptide orpeptide including, but not limited to, a structural protein, an enzyme,a cytokine (such as an interferon and/or an interleukin) an antibiotic,a polyclonal or monoclonal antibody, or an effective part thereof, suchas an Fv fragment, which antibody or part thereof may be natural,synthetic or humanised, a peptide hormone, a receptor, a signallingmolecule or other protein; a nucleic acid, as defined below, including,but not limited to, an oligonucleotide or modified oligonucleotide, anantisense oligonucleotide or modified antisense oligonucleotide, cDNA,genomic DNA, an artificial or natural chromosome (e.g. a yeastartificial chromosome) or a part thereof, RNA, including mRNA, tRNA,rRNA, siRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus orvirus-like particles; a nucleotide or ribonucleotide or syntheticanalogue thereof, which may be modified or unmodified; an amino acid oranalogue thereof, which may be modified or unmodified; a non-peptide(e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate.Small molecules, including inorganic and organic chemicals, which bindto Itch are also included. Examples of small molecules include but arenot limited to small peptides or peptide-like molecules.

Typically, the agent will be an organic compound. Typically the organiccompounds will comprise two or more hydrocarbyl groups. Here, the term“hydrocarbyl group” means a group comprising at least C and H and mayoptionally comprise one or more other suitable substituents. Examples ofsuch substituents may include halo-, alkoxy-, nitro-, an alkyl group, acyclic group etc. In addition to the possibility of the substituentsbeing a cyclic group, a combination of substituents may form a cyclicgroup. If the hydrocarbyl group comprises more than one C then thosecarbons need not necessarily be linked to each other. For example, atleast two of the carbons may be linked via a suitable element or group.Thus, the hydrocarbyl group may contain hetero atoms. Suitable heteroatoms will be apparent to those skilled in the art and include, forinstance, sulphur, nitrogen and oxygen. For some applications,preferably the agent comprises at least one cyclic group. The cyclicgroup may be a polycyclic group, such as a non-fused polycyclic group.For some applications, the agent comprises at least the one of saidcyclic groups linked to another hydrocarbyl group.

The agent may contain halo groups. Here, “halo” includes fluoro, chloro,bromo or iodo.

The agent may contain one or more of alkyl, alkoxy, alkenyl, alkyleneand alkenylene groups—which may be unbranched—or branched-chain.

The agent may be capable of displaying other therapeutic properties.

The agent may be used in combination with one or more otherpharmaceutically active agents—such as one or more otherpharmaceutically active agents that can be used to treat proliferativedisorders such as cancer.

If combinations of active agents are administered, then they may beadministered simultaneously, separately or sequentially.

If the agent is an antibody then it may include but is not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression libraries. Such fragments includefragments of whole antibodies which retain their binding activity for atarget substance, Fv, F(ab′) and F(ab′)₂ fragments, as well as singlechain antibodies (scFv), fusion proteins and other synthetic proteinswhich comprise the antigen-binding site of the antibody. The antibodiesand fragments thereof may be humanised antibodies, for example asdescribed in EP-A-239400. Furthermore, antibodies with fully humanvariable regions (or their fragments), for example, as described in U.S.Pat. Nos. 5,545,807 and 6,075,181 may also be used. Neutralizingantibodies.

Antibodies may be produced by standard techniques, such as byimmunisation or by using a phage display library

An Itch polypeptide or peptide may be used to develop an antibody byknown techniques. Such an antibody may be capable of bindingspecifically to the Itch protein or a homologue, variant, fragment orderivative thereof.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) may be immunised with an immunogeniccomposition comprising an Itch polypeptide or peptide. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminium hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (BacilliCalmette-Guerin) and Corynebacterium parvum are potentially useful humanadjuvants which may be employed if purified the substance amino acidsequence is administered to immunologically compromised individuals forthe purpose of stimulating systemic defence.

Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies to anepitope obtainable from an Itch polypeptide contains antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art. In order that such antibodies may bemade, the invention also provides amino acid sequences of the inventionor fragments thereof haptenised to another amino acid sequence for useas immunogens in animals or humans.

Monoclonal antibodies directed against epitopes obtainable from an Itchpolypeptide or peptide can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus. Panels of monoclonal antibodies produced againstorbit epitopes can be screened for various properties; i.e., for isotypeand epitope affinity.

Monoclonal antibodies may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (1975 Nature 256:495-497),the trioma technique, the human B-cell hybridoma technique (Kosbor et al(1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,779) canbe adapted to produce the substance specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for the Itchpolypeptide or peptide may also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse W D et al (1989) Science256:1275-1281).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies toItch polypeptides. Also, transgenic mice, or other organisms includingother mammals, may be used to express humanized antibodies.

The agent may be an aptamer ie. an oligonucleotide that is capable offorming a complex with an intended target—such as Itch. Aptamers may beprepared by any known method, including synthetic, recombinant, andpurification methods, and may be used alone or in combination with otheraptamers specific for the same target. The oligonucleotides may besingle-stranded, double-stranded, or even triple- or quadruple-strandedstructures. In general, a minimum of approximately 3 nucleotides,preferably at least 5 nucleotides, are necessary to effect specificbinding. Oligonucleotides of sequences shorter than 10 bases may befeasible if the appropriate interaction can be obtained in the contextof the environment in which the target is placed, although aptamers ofabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 nucleotides or more in length are contemplated. Methods ofconstructing and determining the binding characteristics of aptamers arewell known in the art. For example, such techniques are described inU.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459. General teachings onaptamers can be found in Blackwell et al., Science (1990) 250:1104-1110;Blackwell et al., Science (1990) 250:1149-1152; Tuerk & Gold, Science(1990) 249:505-510; and Joyce, Gene (1989) 82:83-87.

Cell

The cell (eg. tissue) that is used in accordance with the presentinvention is a cell that expresses Itch. The cell may be any prokaryoticor eukaryotic cell. Cells that are particularly useful in the context ofthe present invention include mammalian cells and, in particular, humancells of a variety of cell types.

Preferably, the cell is selected from the group consisting of humanembryonic kidney cells such as Hek293, HeLa, MEF, human lung carcinomacells such as H1299 and human osteosarcoma cells such as Saos-2.

A person skilled in the art will appreciate that other cells may also beused in accordance with the present invention—such as host cellsexpressing Itch and/or its substrates including p63 and p73. These cellsmay be particularly useful when using assays or screens to identifyantagonists of Itch. Host cells include any cell that could comprise anItch nucleotide sequence and/or a p63 or p73 nucleotide sequence (or avariant, homologue, fragment or derivative thereof) coding forrecombinant Itch or its substrates, wherein a promoter can allowexpression of the nucleotide sequence when present in the host cell. Thehost cells will be chosen to be compatible with the vector carrying thenucleotide sequences and may for example be prokaryotic or eukaryotic.

Depending on the nature of the polynucleotide encoding the polypeptide,and/or the desirability for further processing of the expressed protein,prokaryotic hosts or eukaryotic hosts—such as yeasts or other fungi—maybe used.

The use of suitable host cells—such as mammalian, yeast, fungal andplant host cells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, proteolytic processing, lipidation andtyrosine, serine or threonine phosphorylation) as may be needed toconfer optimal biological activity on the recombinant expressionproducts.

siRNA

A nucleic acid may be delivered into a cell that modulates the activityand/or expression of Itch, for example, at the level of transcription,transcript stability, translation or post-translational stability. Forexample, the nucleic acid may be an antisense sequence or an siRNA.

The inhibition of gene expression using antisense technology is wellknown. For example, antisense constructs are described in detail in U.S.Pat. No. 6,100,090 (Monia et al), and Neckers et al., 1992, Crit. RevOncog 3(1-2):175-231.

The siRNA may comprise partially purified RNA, substantially pure RNA,synthetic RNA, or recombinantly produced RNA, as well as altered RNAthat differs from naturally-occurring RNA by the addition, deletion,substitution and/or modification of one or more nucleotides.

Such alterations can include the addition of non-nucleotidematerial—such as modified nucleotides—to, for example, the end(s) of thesiRNA or to one or more internal nucleotides of the siRNA, includingmodifications that make the siRNA resistant or even more resistant tonuclease digestion.

Typically, the siRNA will be in the form of isolated siRNA comprisingshort double-stranded RNA from about 17 nucleotides to about 29nucleotides in length—such as approximately 19-25 contiguous nucleotidesin length—that are targeted to a target mRNA. The siRNA comprise a senseRNA strand and a complementary antisense RNA strand annealed together bystandard Watson-Crick base-pairing interactions. The sense strandcomprises a nucleic acid sequence which is identical to a targetsequence contained within the target mRNA.

A target sequence on the target mRNA encoding Itch may be selected froma given sequence—such as a cDNA sequence—corresponding to the targetmRNA, using various methods in the art. For example, the rational designof siRNAs is described in Nat Biotechnol. (2004) 22(3):326-30. SuitablesiRNA molecules are described herein.

Although siRNA silencing is highly effective by selecting a singletarget in the mRNA, it may be desirable to design and employ twoindependent siRNA duplexes to control the specificity of the silencingeffect.

siRNA may be obtained using a number of techniques known to those ofskill in the art. For example, the siRNA may be chemically synthesizedusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesiser. The siRNA may be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions.

siRNA may be recombinantly produced using methods known in the art. Forexample, siRNA may be expressed from recombinant circular or linear DNAplasmids using any suitable promoter. The recombinant plasmids of theinvention can also comprise inducible or regulatable promoters forexpression of the siRNA in a particular tissue or in a particularintracellular environment.

Treatment

As used herein, the term “treatment” includes curing or treatingproliferative disorders including cancer, causing the symptoms of suchdisorders to diminish, and ablating or otherwise alleviating thedisease.

Pharmaceuticals

The agents—such as the Itch antagonists—that modulate (eg. decrease) theactivity and/or expression of Itch will typically be formulated into apharmaceutical composition. In this regard, and in particular for humantherapy, even though the agents described herein can be administeredalone, they will generally be administered in admixture with apharmaceutical carrier, excipient or diluent selected with regard to theintended route of administration and standard pharmaceutical practice.

By way of example, in the pharmaceutical compositions, the agents may beadmixed with any suitable binder(s), lubricant(s), suspending agent(s),coating agent(s), or solubilising agent(s).

Tablets or capsules of the agents may be administered singly or two ormore at a time, as appropriate. It is also possible to administer theagents in sustained release formulations.

Thus, the present invention also provides a method of treatingproliferative disorders in a subject comprising administering to saidsubject an effective amount of an Itch antagonist.

Typically, the pharmaceutical compositions—which may be for human oranimal usage—will comprise any one or more of a pharmaceuticallyacceptable diluent, carrier, excipient or adjuvant. The choice ofpharmaceutical carrier, excipient or diluent can be selected with regardto the intended route of administration and standard pharmaceuticalpractice. As indicated above, the pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

For some embodiments of the present invention, the pharmaceuticalcompositions will comprise an agent that has been screened by the assaymethod(s) described herein.

Such molecules may provide the basis for treatment of proliferativedisorders such as cancer. For example, such molecules may be used toincrease apoptosis.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of recombinant sense or antisense molecules to the targetedcell population. Methods, which are well known to those skilled in theart, can be used to construct recombinant vectors containing thereceptor. Alternatively, the recombinant receptor can be delivered totarget cells in liposomes.

The pharmaceutical composition could be for veterinary (i.e. animal)usage or for human usage.

The pharmaceutical compositions obtained may be useful for preventingand/or treating proliferative disorders such as cancer.

It will be appreciated by those skilled in the art that the agent may bederived from a prodrug. Examples of prodrugs include certain protectedgroup(s) which may not possess pharmacological activity as such, butmay, in certain instances, be administered (such as orally orparenterally) and thereafter metabolised in the body to form an agentthat is pharmacologically active.

It will be further appreciated that certain moieties known as“pro-moieties”, for example as described in “Design of Prodrugs” by H.Bundgaard, Elsevier, 1985, may be placed on appropriate functionalitisof the agents. Such prodrugs are also included within the scope of theinvention.

The agent may be administered as a pharmaceutically acceptable salt.Typically, a pharmaceutically acceptable salt may be readily prepared byusing a desired acid or base, as appropriate. The salt may precipitatefrom solution and be collected by filtration or may be recovered byevaporation of the solvent.

The agent may be prepared by chemical synthesis techniques.

It will be apparent to those skilled in the art that sensitivefunctional groups may need to be protected and deprotected duringsynthesis of a compound of the invention. This may be achieved byconventional techniques, for example, as described in “Protective Groupsin Organic Synthesis” by T W Greene and P G M Wuts, John Wiley and SonsInc. (1991), and by P. J. Kocienski, in “Protecting Groups”, GeorgThieme Verlag (1994).

It is possible during some of the reactions that any stereocentrespresent could, under certain conditions, be racemised, for example, if abase is used in a reaction with a substrate having an having an opticalcentre comprising a base-sensitive group. This is possible during e.g. aguanylation step. It should be possible to circumvent potential problemssuch as this by choice of reaction sequence, conditions, reagents,protection/deprotection regimes, etc. as is well-known in the art.

The compounds and salts may be separated and purified by conventionalmethods.

Separation of diastereomers may be achieved by conventional techniques,e.g. by fractional crystallisation, chromatography or H.P.L.C. of astereoisomeric mixture of a compound of formula (I) or a suitable saltor derivative thereof. An individual enantiomer of a compound of formula(I) may also be prepared from a corresponding optically pureintermediate or by resolution, such as by H.P.L.C. of the correspondingracemate using a suitable chiral support or by fractionalcrystallisation of the diastereomeric salts formed by reaction of thecorresponding racemate with a suitably optically active acid or base.

The agent or variants, homologues, derivatives, fragments or mimeticsthereof may be produced using chemical methods to synthesise the agentin whole or in part. For example, if the agent comprises a peptide, thenthe peptide can be synthesised by solid phase techniques, cleaved fromthe resin, and purified by preparative high performance liquidchromatography (e.g., Creighton (1983) Proteins Structures And MolecularPrinciples, WH Freeman and Co, New York N.Y.). The composition of thesynthetic peptides may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure; Creighton, supra).

Synthesis of peptide agents (or variants, homologues, derivatives,fragments or mimetics thereof) can be performed using varioussolid-phase techniques (Roberge J Y et al (1995) Science 269: 202-204)and automated synthesis may be achieved, for example, using the ABI 43 1A Peptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer. Additionally, the amino acid sequencescomprising the agent, may be altered during direct synthesis and/orcombined using chemical methods with a sequence from other subunits, orany part thereof, to produce a variant agent.

The agent may be a modified agent—such as, but not limited to, achemically modified agent.

The chemical modification of an agent may either enhance or reducehydrogen bonding interaction, charge interaction, hydrophobicinteraction, Van Der Waals interaction or dipole interaction.

In one aspect, the agent may act as a model (for example, a template)for the development of other compounds.

Administration

The term “administered” includes delivery by viral or non-viraltechniques. Viral delivery mechanisms include but are not limited toadenoviral vectors, adeno-associated viral (AAV) vectors, herpes viralvectors, retroviral vectors, lentiviral vectors, and baculoviralvectors. Non-viral delivery mechanisms include lipid mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationic facialamphiphiles (CFAs) and combinations thereof.

The components may be administered alone but will generally beadministered as a pharmaceutical composition—e.g. when the componentsare in admixture with a suitable pharmaceutical excipient, diluent orcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

For example, the components can be administered in the form of tablets,capsules, ovules, elixirs, solutions or suspensions, which may containflavouring or colouring agents, for immediate-, delayed-, modified-,sustained-, pulsed- or controlled-release applications.

If the pharmaceutical is a tablet, then the tablet may containexcipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine, disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscarmellose sodium and certain complex silicates,and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia. Additionally, lubricating agents such asmagnesium stearate, stearic acid, glyceryl behenate and talc may beincluded.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the agent may becombined with various sweetening or flavouring agents, colouring matteror dyes, with emulsifying and/or suspending agents and with diluentssuch as water, ethanol, propylene glycol and glycerin, and combinationsthereof.

The routes for administration (delivery) may include, but are notlimited to, one or more of oral (e.g. as a tablet, capsule, or as aningestable solution), topical, mucosal (e.g. as a nasal spray or aerosolfor inhalation), nasal, parenteral (e.g. by an injectable form),gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, vaginal, epidural, sublingual.

Conveniently, administration may be by inhalation. Commerciallyavailable nebulisers for liquid formulations, including jet nebulisersand ultrasonic nebulisers are useful for such administration. Liquidformulations can be directly nebulised and lyophilised powder can benebulised after reconstitution.

For administration by inhalation, the agents are conveniently deliveredin the form of an aerosol spray presentation from pressurised packs ornebulisers. The agents may also be delivered as powders which may beformulated and the powder composition may be inhaled with the aid of aninsufflation powder inhaler device.

Dose Levels

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

Formulation

The component(s) may be formulated into a pharmaceutical composition,such as by mixing with one or more of a suitable carrier, diluent orexcipient, by using techniques that are known in the art.

Kits

The materials for use in the present invention are ideally suited forthe preparation of kits.

Such a kit may comprise containers, each with one or more of the variousreagents (optionally in concentrated form) utilised in the methods,including, a cell that expresses or is capable of expressing Itch; andone of its substrates including p63 or p73.

The kit optionally further comprises one or more controls—such as a cellthat cannot express Itch.

A set of instructions will also typically be included.

Suitably, the kit is a diagnostic kit for measuring Itch expression intumours. Use of such a kit, for example, will allow treatment diagnosisas Itch expression correlates with therapy insensitivity or inverselycorrelates with survival.

General Recombinant DNA Methodology Techniques

The present invention employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, microbiology, recombinantDNA and immunology, which are within the capabilities of a person ofordinary skill in the art. Such techniques are explained in theliterature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.(1995 and periodic supplements; Current Protocols in Molecular Biology,ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 The Ubiquitin-Protein Ligase Itch Regulates p73Stability

Materials and Methods

Plasmids

Myc-Itch and Myc-Itch MUT plasmids (C830A) were provided by Dr. T.Pawson (Winberg, 2000). Itch GST fusion proteins, were generated bysubcloning PCR fragments into the BamHI and SalI sites of pGEX-6P1(Amersham Pharmacia Biotech). The following fragments were cloned:GST-Itch, from Thr 277 to Glu 903 (lacking the N-terminal C2 domain) andGST-WW, from Pro 317 to Pro 520 (spanning only the four WW domains).HA-p73

and HA-p73

were described previously (De Laurenzi, 1998). GST-p73 fusion proteinencompassing only the PY motif (GST-PY, from Met 452 to Ala 489) wasobtained by subcloning into BainHI and NotI sites of the pGEX-6P1. Thepoly-HA-ubiquitin construct (HA-Ub) was kindly provided by Dr. D.Bohmann (Treier, 1994). pET23a-Ubch7 (E2) bacterial expression vectorsdirecting the synthesis of the E2 enzyme (Ubch7) and the E1ubiquitin-activating enzyme Uba1 were a gift of Dr. P. M. Howley (Kumar,1997). The reporter plasmid containing the luciferase cDNA under controlof the Bax and p21 promoter were provided by Dr. Levrero (Vossio, 2002);that under the control of the MDM2 promoter was provided by Dr. Blandino(Strano, 2001). Nedd4-myc construct was a gift from Dr. D. Rotin. Allp73 mutants were generated by site directed mutagenesis using theQuickchange kit (Stratagene) according to the manufacturer'sinstructions.

Cell Culture and Transfection

Human embryonal kidney cells (Hek293), HeLa cells and MEFs were grown inDulbecco's modified Eagle medium (DMEM) (GibcoBRL); the human lungcarcinoma cells H1299 and the human osteosarcoma cells Saos-2 werecultured in RPMI (GibcoBRL). MEF Itch−/− cells were prepared fromItch−/− mice (Y—C-Liu) or they were generous gift from Neil Copeland,Lynda Matesic, Nancy Jenkins. All media were supplemented with 10% (v/v)fetal bovine serum (FBS) (GibcoBRL). Saos-2-TAp73

were a kind gift of K. Vousden (Nakano, 2000). Saos-2-ΔNp73

were generated as described before (Maisse et al., 2004). For the TET-Oncell lines media was supplemented with 10% (v/v) tetracycline free FBS(Clonetech). Genes were induced by adding 0.5 μg/ml doxycycline. Allcell lines were grown at 37° C. in a humidified atmosphere of 5% (v/v)CO₂ in air. Transient transfection was performed with lipofectamine 2000reagent according to the manufacturer's instructions. Apoptosis wasanalysed by flow cytometric evaluation of DNA fragmentation.

Western Blot and Antibodies

Proteins were separated on SDS-PAGE and blotted onto nitrocellulosemembranes. Filters were blocked with TBST 5% non-fat dry milk andincubated with primary antibodies for 2 hrs at room temperature (RT).Filters were incubated for 1 hour at RT using the appropriatehorseradish peroxidase-conjugated secondary antibody (rabbit and mouseBioRad; goat Santa Cruz). Detection was performed with the enhancedchemiluminescence Supersignal West Pico (Pierce). Anti-p73 is amonoclonal antibody (ER15) (Neomarkers). Endogenous Itch was detectedwith a mouse monoclonal antibody (BD Bioscience), Actin (sc-1615)polyclonal goat antibody (Santa Cruz), Lamin B (sc-6217) goat polyclonalantibody (Santa Cruz), Hsp70 (sc-1060) polyclonal goat antibody (SantaCruz), β-Tubulin (sc-9104) polyclonal rabbit antibody (Santa Cruz), p21(sc-756) polyclonal rabbit antibody (Santa Cruz), p53 DO-1 and Pab1801monoclonal mouse antibodies (Santa Cruz). c-Myc-tagged constructs weredetected or immuno-precipitated with the sc-40 monoclonal mouse antibody(Santa Cruz), HA-tagged with the sc-805 polyclonal rabbit antibody(Santa Cruz), and the Flag-tagged constructs with the M2 monoclonalmouse antibody (Sigma).

Affinity Selection

An aliquot of a T7 phage display library of human brain cDNA (Novagen)was incubated overnight at 4° C., with 50 μg of GST-PY p73α_(.)fusionprotein immobilized on glutathionesepharose beads (AmershamBiosciences), in the presence of 1% bovine serum albumin. Unbound phagewere removed by washing five times with 1 ml of 0.1% Tween 20 in PBS,and then once in 1 ml PBS and resuspended in 50 μl of PBS. The boundphages were recovered by incubating at 37° C. with 2 ml of E. ColiBLT5615 (Novagen) induced with 1 mM IPTG for 30 min before phageaddition. After cell lysis, the phage lysate was clarified bycentrifugation, and used in a new selection round. After three selectioncycles the resulting phages were analyzed by a plaque assay (Zucconi,2001), using soluble purified GST-PY at a concentration of 5 μg/ml.Positive plaques were isolated from plates. 1 μl of the suspension wasamplified by PCR using primers to the regions flanking the insert, andfragments were sequenced. To estimate the enrichment of Itch, 1 μl ofclarified phage lysate corresponding to each of the different selectionrounds, was amplified by PCR (20 amplification cycles).

GST-Fusion Proteins and Pull-Down Assays

GST fusion proteins were expressed in E. Coli BL21 (DE3) and purified onglutathionesepharose beads (Amersham Biosciences) following standardprocedures. For the pull-down assay, Hek293 cells transfected withHA-TAp73α or empty vector, were lysed in 50 mM Tris HCl, pH 8, 150 mMNaCl, 1 mM MgCl₂, 1 mM EGTA, 10% glycerol, 100 mM NaF and 1% TritonX-100 containing protease cocktails (Sigma) and centrifuged toprecipitate cellular debris. 1.5 mg of total cellular proteins werefirst precleared with glutathione-sepharose beads and then incubated for2 hrs. at 4° C. with immobilized GST fusion proteins (25 μg). Unboundproteins were removed by washing four times with 0.1% Tween20 in PBS,and the precipitates were resolved by SDS-PAGE. The immunoblots wereprobed with the indicated antibodies.

Immunoprecipitation

Following a previously published procedure (Gottifredi, 1999) Hek293cells were transiently transfected with 8 μg of total DNA of theindicated mammalian expression plasmids and harvested 48 hours aftertransfection. Cells were then lysed as described above. Followingpreclearing for 1 h at 4° C., we performed immunoprecipitation byincubating 1.5 mg of whole-cell extracts with the indicated antibodies,rocking at 4° C. for 1 hour. The immunocomplexes were collected byincubating with protein G Agarose (KPL), washed with Net-gel buffer (50mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, 0.1% NP40). Thebeads were then resuspended in 5× Laemmli buffer, and subjected towestern blot with the indicated primary antibodies.

Ubiquitination Assays

In vitro assays were performed as described previously (Hamilton, 2001),using in vitro translated radiolabeled p73 and p53 proteins. In vitrotranscription/translation of proteins was performed using the rabbitreticulocyte lysate system TNT kit (Promega), in the presence of[³⁵S]Met (Amersham Biosciences) according to the manufacturer'sprotocol. Ubiquitination reactions mixture contained: 2 μl of E. ColiBL21 bacterial extracts over-expressing wheat E1 and 2 μl of a human E2(UbcH7), 5 μg of purified E3 enzyme (either GST-Itch or GSTItch MUT), 25mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, 2.5 mM ATP, 4 nMMgCl₂, 10 μg of bovine ubiquitin. After incubation for 90 min at 37° C.,the reactions were terminated by adding 5× Laemmli buffer, resolved bySDS-PAGE followed by autoradiography.

For in vivo experiments Hek293 cells were transiently transfected withmammalian expression plasmids for HA-tagged ubiquitin (HA-Ub), with theindicated combination of plasmids. 48 hrs after transfection, cells wereharvested, the insoluble fraction was removed by a high-speed spin, and1 mg of total cellular proteins of the clarified supernatant wassubjected to immuno-precipitation using anti Flag antibodies (Sigma).Ubiquitin-conjugates were detected by Western immuno-blot analysis usingan anti-HA-antibodies (Santa Cruz).

Measurement of p73 Half Life

Decay of p73 protein levels in the presence of cycloheximide.Cycloheximide (20 μg/ml) was added to Hek293 cells 24 hours aftertransfection with a total of 3 μg of the indicated plasmids in a 1:5ratio p73/Itch and p53/Itch. Protein levels were determined bycollecting cells at the indicated time points and performingimmunoblotting as described above. The relative amount of p73 proteinwas evaluated by densitometry and normalized on β-Tubulin. ³⁵S pulsechase. H1299 cells were transfected with a total of 3 μg of theindicated plasmids in a 1:2 ratio p73/Itch. 48 hours post-transfectioncells were starved for 30 minutes in DMEM with dialysed serum and thenlabelled with 250 μCi/ml of Redivue PRO-MIX (L-[³⁵S] in vitro celllabelling mix) for 60 minutes. Unlabeled Met and Cys were added andcells were collected in RIPA buffer (200 mM Tris pH8, 150 mM NaCl, 0.5%Sodium Deoxycholate, 0.1% SDS, 1% NP40 and 0.2 mM EDTA) at the indicatedtimes. Immunoprecipitations were performed with 150 μg of total proteinlysate and 4 μl of anti-HA (Y-11) polyclonal antibody Santa Cruz.Immunoprecipitates were washed six times in RIPA buffer and six times inNETgel and run on a SDS-PAGE and detected by autoradiography.

Steady State Protein Levels Analysis

The concentration of TAp73α, TAp73

and p53 was monitored by Western blotting. 48 hrs after co-transfectionwith HA-TAp73, HA-TAp736 and HA-p53 and either Myc-Itch or Myc-Itch MUT,Hek293 cells were treated for 40 min with or without proteasomeinhibitor MG132 at a final concentration of 50 μM in DMSO. 25 μg of celllysates were subjected to Western blotting. p73 proteins were detectedby an anti-HA antibody. The same blots were re-probed with anti Mycantibody to detect Itch and with anti-Actin antibody to show equalloading.

Promoter Reporter

H1299 cells were transfected with the indicated combinations of plasmidsencoding p73

(10 ng/well of 96-well plate), Myc-Itch or Myc-Itch MUT, or emptycontrol vectors together with the indicated luciferase reporter plasmid(60 ng/well) and Renilla luciferase reporter (1.2 ng/well). The totalamount of transfected DNA in each well was kept constant by the additionof different amounts of empty vector. The luciferase activity wasquantified using a commercially available kit (Dual-Glo, Promega)according to the manufacture's instructions.

Itch siRNA

Saos-2 cells were electroporated with 20 ml of a 20 mM solution oftwenty-one-nucleotide RNA (Qiagen) using a BIO-RAD electroporationapparatus. A mix of two different oligoes was used to downregulate Itch.The Itch target sequences were AAGTGCTTCTCAGAATGATGA andAACCACAACACACGAATTACA and the scrambled sequence wasAATTCTCCGAACGTGTCACGT. Cells were collected after 48 hours and celllysates were subjected to western blot for Itch detection as describedabove.

Results

MDM2, the E3 ubiquitin ligase that regulates the degradation of thecognate protein p53 via a proteasomal dependent pathway, binds to p73but does not promote its degradation (Balint, 1999; Dobbelstein, 1999;Lohrum, 1999; Ongkeko, 1999; Zeng, 1999).

In order to define the degradation pathway of p73 we searched a humancDNA library displayed on bacteriophage capsids for p73 specific bindingpartners. In order to identify mechanisms distinct from those of p53, asa bait we used a p73 C terminal fragment since this region is notpresent in p53 and contains a PpxY (PY) sequence that has beencharacterized as a binding motif for a class of WW domains (FIG. 1A)(Sudol, 1996).

Identification of Itch as a Novel p73 Interacting Partner

To identify new p73 binding proteins, not shared by p53, we fused afragment of TAp73

(Met 452-Ala 489) to GST protein (FIG. 1A) and used it as bait in “phagedisplay” screening (Castagnoli, 2001; Cesareni, 1999). This fragmentcontains a region that is not homologous to p53 and contains aprotein-protein binding motif, known as a PY motif (Strano, 2001) (FIG.1A) characterized by the consensus sequence PPxY. This motif binds to a40 amino-acid long structural domain known as WW domain, organized toform a threestranded, antiparallel Psheet, containing two tryptophan (W)residues, spaced 20-22 amino acids apart (Sudol, 1996). After threerounds of affinity-selection, performed on a human brain cDNA librarydisplayed by a T7 phage vector, we analyzed the resulting phagepopulation by a plaque assay. The affinity selected phage pool containeda high percentage (25%) of positive clones that bound the bait (FIG.1B). By comparing the frequency of positive plaques before and afterselection, we estimated an enrichment of at least 60 fold (FIG. 1B).Several clones contained overlapping protein fragments (all containingthe WW domains) encoded by the Itch gene (FIG. 1A). The enrichment ofclones displaying Itch WW domains was further confirmed by performingPCR reactions with specific oligonucleotides flanking the WW domains(from Pro 317 to Pro 520) (FIG. 1B). In contrast, clones containing theβ-Actin gene were rapidly lost during the selection process (FIG. 1B).

p73 but not p53 Associates with Itch

In order to verify that Itch associates with p73, we performed an invitro pull down assay. Hek293 cells were transfected with either anempty vector or with a vector encoding HA tagged TAp73α. Cell lysateswere mixed separately with a sepharose resin containing either GST orthe WW region of Itch fused to GST (GST-WW). TAp73

was efficiently retained by GST-WW, while no significant binding to GSTalone was detected (FIG. 1C). The interaction was also confirmed byco-immuno-precipitation (co-IP) of overexpressed TAp73

and Itch. As shown in FIG. 1D immuno-precipitation (IP) of Myc taggedItch resulted in co-IP of TAp73α. Addition of proteasome inhibitor MG132resulted in stronger interaction. As expected, since p53 does notcontain the PY motif (FIG. 1A), it did not bind to Itch and could not beprecipitated with Itch regardless of the presence of the proteasomeinhibitor (FIG. 1D). Similarly, TAp73

that also lacks the PY motif (FIG. 1A) could not be co-IP with Itch(data not shown). The N-terminally truncated form ΔNp73

also bound Itch (FIG. 1E). To confirm that the interaction requires thePY motif of p73 we generated mutants of both the PY motifs found in p73(FIG. 1A). FIG. 1F shows that mutants containing the Y487F substitutionlost the ability to bind Itch while the single mutant TAp73α_(Y407F) didnot. In order to confirm that this interaction also occurs in cells atphysiological concentrations, we performed co-IP of endogenous proteins(FIG. 1G). Again IP of endogenous p73 co precipitated Itch, while IP ofp53 did not.

These data clearly demonstrate that endogenous p73

isoforms can bind to the WW domains of Itch through their PY motif andthat the interaction is selective for p73 and not shared by p53.

p73 is Ubiquitinated by Itch

We next investigated whether p73 can serve as a substrate for theubiquitin-protein ligase activity of Itch. We used a recombinant Itch(GST-Itch) (Qiu, 2000), in a reconstituted in vitro ubiquitinationsystem containing Ub, wheat E1, human E2 (UbCH7), ATP, and in vitrosynthesized radio labeled [³⁵S] TAp73

protein as substrate. In the presence of purified GSTItch, the TAp73

protein was ubiquitinated, as shown by the appearance of discrete highermolecular weight TAp73

species (FIG. 2A, lane 1). To demonstrate that the appearance ofubiquitinated forms of TAp73

requires an intact Itch Hect domain, we used a previously describedinactive mutant of Itch (GST-Itch MUT) (C830A) (Winberg, 2000). As shownin FIG. 2A, this mutant, that retains the ability to bind TAp73

(FIG. 1D), lost the ability to promote TAp73

ubiquitination. The inability of Itch to ubiquitinate p738 and p53suggests that the PY motifs are required, since these two proteins lackthese motifs (FIG. 1A and FIG. 2A lanes 4 and 7). These in vitro dataalso show that no other factor was required for this reaction to occur.

We next examined if Itch can catalyze p73 ubiquitination in cells.Extracts of Hek293 cells transfected with plasmids expressing: HA-taggedubiquitin (Ub-HA), Myc-tagged Itch (Myc-Itch) and Flag-tagged TAp73

or p53 (Flag-TAp73

and Flag-p53), were subjected to IP with anti-Flag antibodies anddetected with anti-HA and anti-Flag western blots. As shown in FIG. 2B(lane 5), Ub-HA TAp73

conjugates were detected upon co-transfection only with wild type Itch(Myc-Itch WT), and not with the catalytically inactive mutant of Itch(Myc-Itch MUT) (FIG. 2B, lane 6). Similarly ΔNp73

was ubiquitinated by Itch (FIG. 2C), showing that the N-terminal part ofthe protein is not required for the ubiquitination. In contrast, p53 wasnot ubiquitinated by Itch (FIG. 2B 7-9). Although ubiquitination ofTAp73α_(Y487F) mutant was reduced, a greater reduction was seen with thedouble mutant TAp73α_(Y407F/Y487F) (FIG. 2D). Ubiquitination of theTAp73α_(Y407F) mutant was similar to that of wild type p73. The rightpanel (lanes 10-18) in FIG. 2B demonstrates the specificity of thereaction since no higher molecular weight bands were observed in theabsence of Ub-HA. These data clearly show that p73 but not p53 isubiquitinated by Itch, suggesting that this protein plays an importantrole in the regulation of p73 steady state protein levels.

Itch Regulates the Stability of p73 in Cells

Since ubiquitination of proteins is usually associated with theirturnover (Weissman, 2001), we investigated if Itch can regulate p73protein abundance. We measured TAp73

levels in whole cell extracts in the presence or absence of Itch.Representative data from several independent experiments demonstratethat co-expression of Myc-Itch and TAp73 cc in cells results in astriking decrease of TAp73

levels (FIG. 3A), indicating that the Itch-dependent ubiquitinationtargets p73 for degradation. Consistently, the catalytic mutant of Itchwas not able to alter the concentration of p73 (FIG. 3A, lane 3) and theTAp73α_(Y487F) (FIG. 3B) mutant levels were not affected by Itchover-expression.

Because poly-ubiquitination generally targets proteins for proteasomaldegradation (Weissman, 2001), we determined the effect of MG132 on thesteady state levels of TAp73 cc. As shown in FIG. 3A (lanes 4-6),addition of MG132 to cells blocks the Itch-mediated TAp73α degradationand resulted in the accumulation of the ubiquitinated forms of TAp73

(data not shown). This is consistent with previous reports showing thatproteasome inhibition leads to the stabilization of endogenous p73protein (Balint, 1999). Again TAp736 and p53 levels were not affected byItch (FIG. 3A).

We further confirmed these results by measuring p73 half-life in thepresence or absence of Itch using two different methods. Both pulsechase using ³⁵S labeled Met and Cys (FIG. 3C-D), and cycloheximideblockade (FIG. 3 E-F), showed a marked decrease of TAp73 and ΔNp73 halflives in the presence of Itch. Under similar experimental conditions nochange in p53 half-life was observed (FIG. 3G). To further confirm theimportance of Itch in controlling p73 steady state levels we reducedendogenous Itch levels by siRNA. To this end we used Tet-On inducibleSaos-2 cells expressing p73 (Melino et al., 2004). FIG. 4A shows that,in this inducible cell line, reduction in Itch expression by siRNAresulted in more rapid induction and higher levels of p73 protein.Moreover, after withdrawal of induction, p73 levels decline more slowlywhen Itch expression is reduced (FIG. 4B).

Similarly the steady state levels of endogenous TAp73 and ΔNp73 isoformsincreased in Saos-2 cells when Itch was down regulated (FIG. 4C). Thisconfirms that basal Itch levels are important in controlling basal p73levels. In agreement, endogenous levels of ΔNp73 (which is the onlyisoform detectable in these cells) were increased in mouse embryofibroblasts (MEFs) derived from non-agouti-lethal 18H Itch deficientmice (MEF Itch−/−) (FIG. 4D). Re-introduction of wild type Itch intoMEFs Itch−/− resulted in reduction of endogenous ΔNp73 levels in thesecells (FIG. 4E).

Not all Itch family members had the same effect on p73. Nedd4 bound(FIG. 5A) both TAp73 and ΔNp73 but was not capable of catalyzing theubiquitination of these proteins (FIG. 5C) and therefore did not affecttheir degradation (FIG. 5B). Miyazaki and colleagues (Miyazaki et al.,2003) demonstrated that NEDL2, another Nedd4 family member, binds,ubiquitinates and stabilizes p73. Thus, different Nedd4-like E3 ligases,although all capable of binding the p73 PY motifs, exert differenteffects on these proteins.

Itch Decreases p73-Dependent Transcriptional Activity

To evaluate if the interaction between p73 and Itch influences itstranscriptional activity, we co-transfected H1299 cells with TAp73

and Myc-Itch or Myc-Itch MUT and assessed TAp73

transcription activity by luciferase reporter assay using differentp53/p73 responsive promoters (Bax, p21 and MDM2). As shown in FIG. 6(A-C-E), consistent with a reduction in TAp73 protein levels,co-transfection of Myc-Itch reduced the transcriptional activity ofTAp73

on all the promoters tested in a dose dependent manner. As expected themutated form of Itch had no effect on the transcriptional activity ofp73 (FIG. 6B-D-F). The reduction of the promoter activity was paralleledby a reduction in endogenous levels of p73 target proteins such as p21(FIG. 6G).

Itch is Down Regulated in Response to DNA Damage

Since TAp73 protein levels increase in response to DNA damage (Agami,1999; Gong, 1999; Yuan, 1999) we investigated if Itch expression is alsomodulated after DNA damage. In Saos-2 cells, following treatment withdoxorubicin, to induce DNA damage, endogenous Itch protein levels weredown-regulated in a time (FIG. 7A) and dose (not shown) dependentmanner. The reduction of Itch levels was paralleled by an increase inendogenous TAp73 levels (FIG. 7A). Reduction of Itch also paralleled anincrease in apoptosis (FIG. 7B). This response of Itch to doxorubicintreatment is not cell type specific: FIG. 7C shows a similar experimentusing HeLa, H1299 and Hek293 cell lines treated with doxorubicin. Theseresults suggest that Itch could be an important regulator of TAp73levels and that upon DNA damage Itch is down regulated allowing TAp73levels to rise. FIG. 9 shows that down-regulation of Itch by siRNAsensitizes cells to doxorubicin.

This pathway is p53 independent since the effect on cell cycle andapoptosis was also observed in cell lines lacking p53 such as Saos-2.The DNA damage-dependent decrease in Itch levels is not specific fordoxorubicin treatment but also occurs when cells are treated with otherDNA damaging agents. As shown in FIG. 7D treatment with cisplatin andetoposide also resulted in a dramatic reduction in Itch protein levels.Since Itch also affects ΔNp73 levels an increase of this isoform wouldalso be expected in response to DNA damage. FIG. 7E, however, shows thatas we have previously published (Maisse et al., 2004), ΔNp73

unlike TA is rapidly degraded upon DNA damage regardless of thedown-regulation of Itch. In order to confirm that ΔNp73 degradation isindependent of Itch we reduced Itch levels in the Saos-2-ΔNp73 ccinducible cell line, and then induced ΔNp73 expression by the additionof doxycycline. The inducer was removed and DNA damage was then inducedwith doxorubicin. As shown in FIG. 7F reduction of Itch levels resultsin increased stabilization of ΔNp73 cc but does not prevent itsdegradation in response to doxorubicin treatment.

Discussion

p73 activity depends on its steady state protein levels and a variety ofevidence suggests that post-transcriptional regulation rather thantranscriptional control plays a major role in p73 response to DNAdamage. Upon DNA damage, p73 becomes activated and stabilized (Catani,2002), and this results in cell cycle arrest and apoptosis (Agami, 1999;Gong, 1999; Yuan, 1999). p73 is rarely mutated in cancers, althoughaltered levels of one or more isoforms of p73 have been found in tumors(Casciano, 2002; Ikawa, 1999; Melino, 2002; Putzer, 2003; Romani, 2003;Tschan, 2000). Little is known about the pathways leading to p73degradation, despite the likely importance of its consequences in cancerdevelopment and therapy.

Endogenous p73 steady state levels increase in the presence ofproteasome inhibitors, suggesting a role for this pathway in p73degradation (Balint, 1999; Ongkeko, 1999; Zeng, 1999). While thehomology with p53 suggests that Ub-protein ligase MDM2 could be therequired E3 ligase, MDM2 binding to p73 does not lead to p73ubiquitination and degradation but instead leads to p73 stabilization(Balint, 1999; Dobbelstein, 1999; Lohrum, 1999; Ongkeko, 1999; Zeng,1999).

We have recently reported that, upon activation of p38 MAP kinase, p73is recruited into the PML-NB and acetylated by p300. Acetylationprotects TAp73 from ubiquitination resulting in its stabilization(Bernassola et al., 2004). NEDL2 a HECT-type E3 ligase has also beenreported to bind and ubiquitinate p73 resulting in its stabilizationrather than degradation (Miyazaki et al., 2003). Regulation of p73stability, and particularly that of its different isoforms, is thereforecomplex and far from fully understood.

Here, we demonstrate that Itch selectively binds and ubiquitinates p73C: In contrast, it has no such effect on p53. Our results also show adifferent regulation among the various C-terminal isoforms of p73 sinceonly the

and

isoforms that contain the PY motif are regulated by Itch while the δ and

isoforms that lack this domain escape this regulation. We found thatItch is rapidly down-regulated in response to DNA damage allowing p73levels to increase. Therefore, this response to DNA damage, selectivefor p73 and independent of p53, may be important in p53 negative tumors.This is supported by descriptions, for example, in Cell, 2004,119(6):861-72 and Cell, 119(6):847-60.

We have recently shown that unlike TAp73, the ΔNp73 isoforms are rapidlydegraded in response to DNA damage (Maisse et al., 2004). Our resultsshow that ΔNp73 isoforms are also Itch substrates and Itchdown-regulation results in increased ΔNp73 levels. The down-regulationof Itch in response to DNA damage should therefore result in a parallelincrease of TAp73 and ΔNp73 forms. This however is not the case andwhile TAp73 levels actually rise, ΔNp73 is rapidly degraded (Maisse etal., 2004). In our model, Itch seems to be responsible for keeping bothTAp73 and ΔNp73 levels low under normal condition (FIG. 8). Upon DNAdamage, Itch reduction allows stabilization of both TAp73 and ΔNp73.Therefore, a second pathway should specifically target ΔNp73 fordegradation. Recently Toh et al. describe the ability of c-Jun toregulate the stability of p73 (Toh et al., 2004). Even though theunderlying molecular mechanism was not elucidated, here c-Jun affectsstability of TAp73 and not ΔNp73, indicating a differential effect onthe two major p73 isoforms. In conclusion we provide a mechanism bywhich p73 levels are normally kept low in cells, in accordance with thelow basal levels found in many tissues and cell lines, and are increasedin response to DNA damage (FIG. 8).

Example 2 An Interaction Between p63 and ITCH

Methods

Plasmids

Myc-Itch and Myc-Itch MUT plasmids (C830A) were provided by Dr. T.Pawson. Flag-TAp63α and Flag-ΔNp63α were obtained by subcloning the cDNATAp63α and ΔNp63α into NheI and NotI sites of the pCDNA3.1. TheHA-ubiquitin construct (HA-Ub) was kindly provided by Dr. D. Bohmann.

Cell Culture and Transfection

Human embryonal kidney cells (Hek293), HeLa cells and MEFs were grown inDulbecco's modified Eagle medium (DMEM) (GibcoBRL); the human lungcarcinoma cells H1299 and the human osteosarcoma cells Saos-2 werecultured in RPMI (GibcoBRL). Itch−/− MEF cells were prepared fromItch^(−/−) mice (Y-C-Liu) or they were generous gift from Neil Copeland,Lynda Matesic, Nancy Jenkins. All media were supplemented with 10% (v/v)fetal bovine serum (FBS) (GibcoBRL). All cell lines were grown at 37° C.in a humidified atmosphere of 5% (v/v) CO₂ in air. Transienttransfection was performed with lipofectamine 2000 reagent according tothe manufacturer's instructions. Apoptosis was analysed by flowcytometric evaluation of DNA fragmentation.

Western Blot and Antibodies

Proteins were separated on SDS-PAGE and blotted onto nitrocellulosemembranes. Filters were blocked with TBST 5% non-fat dry milk andincubated with primary antibodies for 2 hrs at room temperature (RT).Filters were incubated for 1 hour at RT using the appropriatehorseradish peroxidase-conjugated secondary antibody (rabbit and mouseBioRad; goat Santa Cruz). Detection was performed with the enhancedchemiluminescence Supersignal West Pico (Pierce). Endogenous Itch wasdetected with a mouse monoclonal antibody (BD Bioscience), Actin(sc-1615) polyclonal goat antibody (Santa Cruz), β-Tubulin (sc-9104)polyclonal rabbit antibody (Santa Cruz), c-Myc-tagged constructs weredetected or immuno-precipitated with the sc-40 monoclonal mouse antibody(Santa Cruz) and the Flag-tagged constructs with the M2 monoclonal mouseantibody (Sigma).

Immunoprecipitation

Following a previously published procedure (Gottifredi, 1999) Hek293cells were transiently transfected with 8 μg of total DNA of theindicated mammalian expression plasmids and harvested 48 hours aftertransfection. Cells were then lysed as described above. Followingpreclearing for 1 h at 4° C., we performed immunoprecipitation byincubating 1.5 mg of whole-cell extracts with the indicated antibodies,rocking at 4° C. for 1 hour. The immuno-complexes were collected byincubating with protein G Agarose (KPL), washed with Net-gel buffer (50mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, 0.1% NP40). Thebeads were then resuspended in 5× Laemmli buffer, and subjected towestern blot with the indicated primary antibodies.

Ubiquitination Assays

Hek293 cells were transiently transfected with mammalian expressionplasmids for HA-tagged ubiquitin (HA-Ub), with the indicated combinationof plasmids. 48 hrs after transfection, cells were harvested, theinsoluble fraction was removed by a high-speed spin, and 1 mg of totalcellular proteins of the clarified supernatant was subjected toimmunoprecipitation using anti Flag antibodies (Sigma).Ubiquitin-conjugates were detected by Western immunoblot analysis usinganti-HA-antibodies (Santa Cruz).

Measurement of p63 Half Life

Decay of p63 protein levels in the presence of cycloheximide.Cycloheximide (20 μg/ml) was added to Hek293 cells 24 hours aftertransfection with a total of 3 μg of the indicated plasmids in a 1:5ratio TAp63/Itch and ΔNp63/Itch. Protein levels were determined bycollecting cells at the indicated time points and performingimmunobloffing as described above. The relative amount of p63 proteinwas evaluated by densitometry and normalized on β-Tubulin.

Steady State Protein Levels Analysis

Levels of TAp63α αν

ΔNp63α were determined 48 hrs after co-transfection with HA-TAp63 andHA-ΔNp63α, in the presence or absence of Myc-Itch or Myc-Itch MUT. 25 μgof cell lysates were subjected to Western blotting. p63 proteins weredetected using an anti-HA antibody. The same blots were re-probed withanti-Myc antibody to detect Itch and with anti-Tubulin antibody asloading control.

Results and Discussion

ITCH Interacts with p63α.

p63α, as well as p73α, has a proline-rich region at its C-terminus,which is present in both the TA and ΔN isoforms and is known to interactwith WW domains, such as those present in ITCH. We tested if ITCHinteracts with Tap63α and ΔNp63α proteins by performingco-immunoprecipitation (co-IP) experiments in 293T cells. We found thatICTH predominantly interacts with the ΔNp63α isoform (FIG. 10). We arecurrently testing whether mutation of the proline-rich domain results inthe loss of binding to ITCH. Previous studies showed that the TAtransactivation domain of p63α is likely to be involved inintramolecular interactions that can mask C-terminal sequences. Thus, itis conceivable that the proline-rich region of p63α is more accessiblein ΔN isoforms, thus allowing the interaction with proteins containing aWW domain, such as ITCH.

ICTH Ubiquitinates p63α.

As ITCH interacts with p63α, we next assessed whether p63α can serve asa substrate of the ubiquitin ligase activity of ITCH. To this end, weoverexpressed ITCH together with TAp63α and ΔNp63α and HA-taggedubiquitin and analyzed the ubiquitination levels of p63 (FIG. 11). Asexpected based on the results of the coIP experiments, only ΔNp63αubiquitination was markedly induced by ITCH (FIG. 11), thusdemonstrating the functional consequences of ITCH/p63 interaction.

p63α Steady State Levels and Half-Life are Affected by ITCH.

In order to determine whether ITCH-dependent ubiquitination of p63αpromotes its downregulation, we measured steady state levels of p63isoforms in the presence or absence of ITCH. While TAp63α levels werenot significantly affected by ITCH, ΔNp63α protein levels were markedlyreduced in cells expressing ITCH (FIG. 12). To prove that the reducedlevels were due to increased degradation, we analyzed the half-life ofboth TAp63α and ΔNp63α in cells expressing ITCH (FIG. 13). In agreementwith the observed decrease in steady state levels of ΔNp63α by ITCH, thehalf-life of ΔNp63α was significantly reduced in the presence of ITCH(FIG. 13A), while the half-life of TAp63α was only marginally affected(FIG. 13B).

In conclusion, our findings clearly show that p63, like p73, is a targetof ITCH-dependent ubiquitination and subsequent degradation. While bothTAp63α and ΔNp63α isoforms are regulated by ITCH, mainly the ΔNp63αisoform serves as a substrate of ITCH. p63 is expressed in the stem cellcompartment of many different epithelia. This suggests that ITCH couldplay a fundamental role in the differential regulation of p63 isoformsduring epithelial differentiation, therefore implying that it could beinstrumental in the restriction of p63 expression to the stem cellcompartment.

Moreover, the observation that Itch selectively targets ΔNp63 forubiquitination and degradation is relevant to epithelial cancers. Asdiscussed above, ΔNp63 expression is increased in the common prostateand breast carcinomas, suggesting that its selective degradation iscompromised. Under these circumstances, therefore, we may be looking forcompounds which either activate Itch, or induce alternative selectiveubiquitination pathways for ΔNp63.

Recent data have implied that disruptions in the balance between TAp63and ΔNp63 isoforms in epithelial tissues may be of greater importance intumorigenesis than direct gene mutation (Park et al., 2000). ΔNp63isoforms may indeed confer a proliferative advantage on cancer cells bycounteracting the transactivation activities of p53 and TAp63 proteinsand, hence, their ability to induce cell cycle arrest and apoptosis(Crook et al., 2000). Interestingly, ΔNp63 is the most highly expressedisoform in squamous cell, prostate and breast carcinomas (Nylander etal., 2002). In addition, loss or impaired expression of TAp63, possiblycaused by altered proteasome-dependent degradation, has been associatedwith tumour progression and poor prognosis in most invasive bladdercancers, which, conversely, display concomitantly up-regulation of ΔNp63(Urist et al., 2002). Hence, the relative up-regulation of ΔNp63 vsTAp63 isoforms in cancers may contribute to promote tumour growth andchemotherapy resistance. Interestingly, loss or reduction of PML proteinexpression has been found in human cancers of various histologic originsincluding epithelial tumours such as prostate and breast carcinomas, inwhich it was associated with tumour grade and progression (Gurrieri etal., 2004). The ability of PML to increase the stability andtranscriptional activity of p63 as well as of the other family membersprovides an explanation for how loss of PML protein would favour tumourinitiation and enforces the notion that p63 may contribute totumorigenesis.

Example 3 Effects of Down-Regulation of Itch in Cancer Cell Lines

Materials and Methods

Cell Culture and Transfections

H1299 and Saos-2 were grown in RPMI medium (GibcoBRL 31870), and HeLa,MCF7, HEK293, HEK293T, A549, A431, Cos-1, cells were grown in Dulbecco'smodified Eagle's medium (GibcoBRL 61965), U2OS and HCT116 were grown inMcCoy's 5A media (GibcoBRL), SH-SY5Y were grown in 50% F12/50% DMEM(Gibco 31330). All media were supplemented with 10% (vol/vol) fetalbovine serum (GibcoBRL), and cells were cultured at 37° C. in ahumidified atmosphere of 5% (vol/vol) CO2 in air. Transienttransfections were performed with Lipofectamine 2000 or CalciumPhosphate reagents according to the protocol of the manufacturer(Invitrogen).

Plasmids

The pSUPER-ITCH2, pSUPER-ITCH18 and pSUPER-scrambled vectors weregenerated by insertion in pSUPER vector (OligoEngine) of oligostargeting the following sequences:

ITCH 2, AAACATTAAAGTCAAACAATATG, ITCH 18 AAGGAGCAACATCTGGATTAATA,(These sequences are 100% identical both in human and mouse ITCH); and ascrambled shRNA control.Western Blot

Samples were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidenedifluoride (PVDF) membranes by means of a semidry blotter. The membraneswere blocked with 5% nonfat dry milk powder in Tris-bufferedsaline-0.05% Tween 20 for 1 h. Immunodetection was performed byincubating the membranes with the different primary antibodies dilutedin blocking buffer for 2 h at room temperature or overnight at 4° C.After four washes with Tris-buffered saline-0.05% Tween 20, themembranes were incubated with secondary antibody conjugated withhorseradish peroxidase for 1 h. After four washes, blots were developedwith ECL Plus detection kit (Amersham), and membranes were exposed toHyperfilm chemiluminescene film (Amersham). The following antibodieswere used: Itch (611199 BD), p73 SAM domain and DN specific antibodies(Sayan et al; 2005), anti-actin (C-11; Santa Cruz), anti-β-tubulin(H-235; Santa Cruz), anti-lamin B (M-20; Santa Cruz)

Effects of shRNA Against Itch on Response to DNA Damage and Effect onp73 Levels

Cells were transfected with plasmid, 24 hours later cells were replatedin 100 mm dishes after a further 24 hours cells were treated withchemotherapeutic agents and samples were collected 0, 24 and 48 hoursafter drug treatment. Samples were collected for protein and FACSanalysis

Cell Cycle Determination

Cells were transfected with a 5:1 ratio of pSUPER-ITCH-2, pSUPER-ITCH-18or pSUPER scrambled together with a plasmid expressing GFP-spectrin.Cell cycle was analysed by flow cytometric evaluation of DNA stainedwith propidium iodide. Cells were fixed in ice cold 70% ethanol andstored at −20° C. Fixed cells were pelletted and resuspended in 50 ulRNAse and incubated at room temperature for 20 minutes. 200 ul ofpropidium iodide (50 ug/ml) was added and cells were incubated furtherat room temperature for 20 minutes. Cell cycle was measured on a FACScan(BD) cytometer using the Cell Quest Program (BD). Cells were gated forGFP expression to allow analysis only of transfected cells and twentythousand events were evaluated using the Cell Quest Program (BD) andModFit LT software (Verity Software).

Results and Discussion

Itch Expression Levels in Human Cell Lines and shRNA Against ITCH

To identify cell lines expressing Itch, lysates from an initial panel of12 cell lines were separated by SDS-PAGE and itch expression wasmeasured by western blot (FIG. 14A). As can be seen in FIG. 14A all celllines tested expressed Itch. Itch was also found to be expressed inprimary mouse embryonic fibroblasts (MEFs—data not shown).

In order to assess the effects of Itch knockdown on the response ofcells to DNA damage, we undertook an RNAi approach to silence Itchexpression. 10 shRNA-expressing vectors were tested for silencingefficiency. Two shRNA expressing vectors (2 and 18) were selected as themost efficient and used for subsequent studies. The effect of theseshRNA vectors on endogenous expression of Itch was determined by westernblot in the cells lines tested in FIG. 14A, and the greatest knockdownwas found to be in Cos1, H1299, HeLa and HEK293T (FIG. 14B). Theknockdown in these cell lines may have be greatest due to highertransfection efficiency in these cell lines with the methods used.

Effect of Itch shRNA on Cell Cycle

To investigate the effects of Itch knockdown on the cell cycle, of thefour different cell lines were transfected with the shRNA vectors forItch and a scrambled sequence, and 48 hours after transfection sampleswere collected and analysed with propidium iodide using a FACScancytometer (FIG. 15). The distribution of the cells in the three stagesof the cell cycle, G1, S-phase and G2, was further analysed using ModFitsoftware (Verity Software) (Table 1). Although a minor increase inpercentage of cells in the G2 phase of cell cycle was observed in H1299cells, overall Itch silencing has little or no effect on the cell cycleof these four cell lines.

TABLE 1 Distribution of cells in G1, G2 and S-phase proportion of cellsafter transfection of scrambled or shRNA ITCH transfection Cell lineshRNA transfected % G1 % S % G2 Cos1 scrambled 36.6 38.6 24.8 Cos1 shRNAItch 2 37.5 36.9 25.6 Cos1 shRNA Itch 18 37.8 34.6 27.6 H1299 scrambled50.7 41.1 8.2 H1299 shRNA Itch 2 45.4 40.1 14.5 H1299 shRNA Itch 1848.12 36.1 15.8 HeLa scrambled 57.6 33.4 8.9 HeLa shRNA Itch 2 60.3 30.09.7 HeLa shRNA Itch 18 57.2 27.5 15.3 HEK293T scrambled 37.1 57.2 5.7HEK293T shRNA Itch 2 44.2 49.2 6.5 HEK293T shRNA Itch 18 45.7 48.8 5.5Effect of Itch shRNA on p73 Levels in Cell Lines

Itch and p73 levels were assessed by western blot in Itch-depleted celllines (FIG. 16). As can be seen in FIG. 16, in Itch-depleted Cos1 cellsp73 protein levels are increased over control levels, a finding which isconsistent with our previous published findings (Rossi et al; 2005). InH1299 cells there is also a possible increase in p73 levels although notas marked as in Cos1.

Effect of Itch shRNA on p73 Levels in Response to DNA Damage

The effect of Itch downregulation upon treatment with cisplatin,etoposide and doxorubicin (DNA-damaging chemotherapeutic agents) hasbeen tested. Remarkably, in H1299 cells, Itch downregulation correlateswith increased p73 levels, thus suggesting that Itch inactivation canpotentiate the effects of chemotherapy by inducing p73 levels (FIG. 17).

Example 4 This Example Demonstrates an Automated High ThroughputEnzyme-Linked Immunosorbent Assay (ELISA) for Use in Screening forInhibitors of Itch

Materials and Methods

Purified GST-Itch and Itch Mutant

Glutathione-S-transferase (GST)-tagged Itch protein and inactivated Itchmutant (C830A) were expressed in bacteria. E. coli BL21CodonPlus®(DE3)-RIL cells (Stratagene) were transformed with either wildtype or mutant constructs prepared in the expression vector pGEX-6PI(Amersham Biosciences). Both GST fusion constructs are comprised of ItchThr 277 to Glu 903, which lack the N-terminal C2 domain. Saturatedcultures were prepared by inoculation of LB medium containing ampicillin(LB/amp) with growth overnight at 37° C. For expression, overnightcultures were diluted 1/100 in LB/amp at 37° C. until they reached an ODof 0.40. At this point the temperature was reduced to 15° C. and IPTG(50 μM final concentration) added to induce expression for 3-4 hourswith shaking at 250 rpm. Cell lystates were prepared and the GST fusionproteins were purified on glutathione-sepharose beads (AmershamBiosciences) using standard procedures.

E1 and E2

EB ubiquitin activating enzyme was expressed in the Baculovirus systemand purified by Ni-NTA chromatography (Qiagen) according to themanufacturer's instructions. The pET23aUbch7 bacterial constructdirecting the synthesis of the E2 ubiquitin conjugating enzyme Ubch7 wasa gift of Dr P M Howley (Kumar S, Kao W H, Howley P M. (1997). Physicalinteraction between specific E2 and Hect E3 enzymes determinesfunctional cooperativity. J Biol. Chem. 272:13548-54). Followingexpression in E. coli BL21 CodonPlus®(DE3)-RIL cells (Stratagene),purified E2 was obtained by Ni-NTA affinity chromatography (Qiagen)according to the manufacturer's instructions.

FLAG-Tagged Ubiquitin

Purified recombinant ubiquitin containing an amino terminal FLAGsequence (DYKDDDDK) was purchased from Sigma Aldrich (cat. No. U5382).

Anti-FLAG M2 Peroxidase Conjugate

The anti-FLAG M2 antibody conjugated to hydrogen peroxidase waspurchased from Sigma Aldrich (cat. No. A8592).

TMB ELISA Substrate

The peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB), wasprepared by making a 1% w/v TMB stock (Sigma Aldrich T-2885) in DMSO anddiluting 1/10 into 0.1M sodium acetate buffer pH 4.5 containing 0.01%v/v hydrogen peroxide just prior to use.

High Throughput Screening Assay for Inhibitors of Itch

The assay was performed using automated liquid handling procedures inorder to test it against the LOPAC¹²⁸⁰™ compound library (SigmaAldrich), which consists of 1280 pharmacologically active compounds.

GST-tagged Itch (350 ng/ml in phosphate buffered saline containing 0.1%Tween 20 (PBST), 20 μl per well) was bound to glutathione-coated,clear-bottomed 384-well ELISA plates (Pierce, custom batch) at 22° C.for 1 hour. Negative control wells assigned to columns 23 and 24 werecoated with an equivalent amount of Itch mutant (C830A). During thisincubation, E2 (UbCH7) was pre-charged with ubiquitin in a 2×concentrated bulk mixture consisting of 468 ng/ml E1, 51 μg/ml E2 and 2μM FLAG-ubiquitin in 1× ubiquitination buffer (25 mM TRIS pH8.0, 100 mMNaCl, 4 mM MgCl₂ 1.25 mM adenosine triphosphate (ATP), 50 μMdithiothreitol (DTT)) for 45 minutes at 22° C. Using this procedure E2was pre-charged with FLAG-ubiquitin before mixing with the testcompounds. At the end of the incubation, the plates were washed 3× withPBST.

The LOPAC¹²⁸⁰™ library compounds were pre-diluted from 10 mM stockplates in dimethylsulfoxide (DMSO) into ubiquitination buffer to give afinal concentration of 10 μM in the assay. Following addition of 10 μlof diluted compound (or equivalent dilution of DMSO to control wells),10 μl of the 2× pre-charge mixture was added to all wells and mixed toinitiate the reaction. The plates were then sealed and theubiquitination reactions allowed to proceed for 2 hours at 22° C. Tostop the reactions and to remove unbound assay components, the plateswere washed 3× with PBST prior to the addition of 20 μl/well anti-FLAGM2 peroxidase conjugate diluted 1/10,000 in PBST. After a furtherincubation of 1 hour at 22° C., the plates were washed 5× and 20 μl TMBsubstrate solution was added to all wells. The substrate development wasallowed to proceed for 15 min at 22° C. before the reactions werestopped with 5 μl per well 1M HCl. The optical density at 450 nm (OD₄₅₀)was determined using a Tecan Safire II automated plate reader. The OD₄₅₀signal is proportional to the amount of FLAG-ubiquitin ligated to theimmobilised GST-Itch protein.

Results and Discussion

To demonstrate that the ELISA assay is specific for ubiquitinated Itchand is dependent on the presence of all the ubiquitination reactioncomponents, the assay was performed with additional controls (FIG. 18).In the presence of E1, E2, FLAG Ub and immobilised wild type Itch, anassay OD₄₅₀ signal of 1.972 was obtained. Under the same conditions butwith substitution of wild type Itch for the mutant Itch (C830A), abackground signal of 0.093 was obtained, indicating a signal:backgroundratio of 21:1 for this assay. The additional controls showed that thesignal was dependent on the presence of wild type Itch together with E1,E2 and FLAG Ub. The omission of any of these components resulted inbackground ELISA signals.

In order to test the performance of a high throughput screening assayfor inhibitors of Itch, a screen of 1280 drug-like small molecules wasperformed using an automated ELISA assay. As an indication of assayperformance and robustness we have applied the Z′ and Z statistics,which are widely quoted for high throughput screening data by theindustry (Zhang J H, Chung T D, Oldenburg K R. (1999). A SimpleStatistical Parameter for Use in Evaluation and Validation of HighThroughput Screening Assays. J Biomol Screen. 4:67-73). For this screenwe obtained average values of 0.76 (range 0.71 to 0.83) and 0.73 (range0.71 to 0.75) for Z′ and for Z respectively. Z′ and Z values in excessof 0.5 are indicative of an excellent assay capable of identifyingoutliers (“hits”) for further investigation and optimisation.

The data obtained across the full screening run is shown in FIG. 19. Thepercentage activity values were calculated using the formula (OD₄₅₀test−mean OD₄₅₀ negative controls)/(mean OD₄₅₀ positive controls−meanOD₄₅₀ negative controls)×100. Wells recording 70% or lower activity(>30% inhibition) were identified as hits. In this screen of 1280compounds, 3 hits were obtained corresponding to an overall rate of0.23%. Therefore a screen of a larger compound library of comparablechemical diversity would be expected to generate a sufficient number ofhits to initiate a study of structure activity relationship (SAR). Theidentification of a SAR is important for the initiation of a medicinalchemistry program at the start of the drug discovery process. In thisscreen the compounds were tested at a fixed concentration of 10 μM,however it will be understood by those skilled in the art that thisconcentration may be increased in order to identify less potentcompounds or reduced in order to identify only those hits with thegreatest inhibitory activity.

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All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

The invention claimed is:
 1. A method of inducing apoptosis in a cellcomprising the step of decreasing the functional activity of Itchpolypeptide or the nucleic acid encoding it, wherein said step comprisescontacting said cell with a composition comprising siRNA or shRNA toItch in an amount effective to induce apoptosis.
 2. A method as claimedin claim 1 wherein altered the functional activity of Itch polypeptideor the nucleic acid encoding it, is inhibition of Itch.
 3. A method forsensitising cells to DNA damaging agents through inhibition of Itchactivity, wherein said inhibition of Itch activity comprises contactingsaid cell with a composition comprising siRNA or shRNA to Itch in anamount effective to sensitize cells to DNA damaging agents.
 4. A methodof decreasing the p73 or p63 ubiquitination activity of Itch in cells ofa subject comprising administering to said subject an agent in an amounteffective to inhibit p73 or p63 ubiquitination activity of Itchsimultaneously or sequentially with a DNA damaging agent, wherein saidagent that inhibits Itch activity is an siRNA or shRNA to Itch, andfurther wherein said subject is a subject that has cancer.
 5. A methodof modulating p63 or p73 stability in a cell comprising modulating Itchactivity or expression, wherein said modulating comprises contactingsaid cell with a composition comprising siRNA or shRNA to Itch in anamount effective to modulate p73 or p63 stability.
 6. A method fordecreasing the activity of Itch in a cell, comprising administering tothe cell an agent in an amount effective to decrease activity of Itchwherein said agent is an siRNA or shRNA to Itch.
 7. A method ofdecreasing the p73 or p63 ubiquitination activity of Itch in the cellsof a subject comprising administering to said subject, in atherapeutically effective amount to decrease the p73 or p63ubiquitination activity of Itch, an siRNA or shRNA to Itch, and furtherwherein said subject is a subject that has cancer.
 8. A method ofdecreasing the p73 or p63 ubiquitination activity of Itch in the cellsof a subject comprising administering to said subject, in atherapeutically effective amount to decrease the p73 or p63ubiquitination activity of Itch, an siRNA or shRNA to Itch, and furtherwherein said subject is a subject that has an epidermal developmentdisorders or immune system dysfunction.
 9. A method of increasingsensitivity of a tumour cell to a chemotherapeutic agent, comprisingreducing Itch activity, expression levels, or function, by contactingsaid cell with a composition comprising siRNA or shRNA to Itch in anamount effective to increase sensitivity of the tumour cell to thechemotherapeutic agent.
 10. The method of claim 1, 4, 6, 7 or 8 whereinsaid agent is an siRNA against Itch.
 11. The method of claim 10, whereinsaid siRNA is further combined with a cytotoxic agent.
 12. The method ofclaim 11, wherein said cytotoxic agent is a DNA damaging agent.
 13. Themethod of any of claim 1, 4, 6, 7 or 8, wherein said siRNA comprises thesequence AAGTGCTTCTCAGAATGATGA (SEQ ID NO:1).
 14. The method of any ofclaim 1, 4, 6, 7 or 8, wherein aid siRNA comprises the sequenceAACCACAACACACGAATTACA (SEQ ID NO:2).